Sensors employing single-walled carbon nanotubes

ABSTRACT

Sensing compositions, sensing element, sensing systems and sensing devices for the detection and/or quantitation of one or more analytes, Compositions comprising carbon nanotubes in which the carbon nanotubes retain their ability to luminesce and in which that luminescence is rendered selectively sensitive to the presence of an analyte. Compositions comprising individually dispersed carbon nanotubes, which are electronically isolated from other carbon nanotubes, yet which are associated with chemical selective species, such as polymers, particularly biological polymers, for example proteins, which can interact selectively with, or more specifically selectivity bind to, an analyte of interest. Chemically selective species bind, preferably non-covalently, to the carbon nanotube and function to provide for analyte selectivity. Chemically selective species include polymers to which one or more chemically selective groups are covalently attached. Chemically selective polymers include, for example, proteins and polysaccharides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application takes priority from U.S. Provisional Patent ApplicationSer. No. 60/590,865 filed Jul. 22, 2004, which is incorporated byreference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made through funding from the United Statesgovernment under National Science Foundation grant number CTS-0330350.The United States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Carbon nanotubes (1, 2) and semi-conducting nanowires (3) holdparticular advantage in sensor applications because their 1-D electronicstructure renders electron transport more sensitive to scattering fromadsorbates than intrinsic mechanisms (2, 4, 5). Hence, these materialshave spawned a host of new conductimetric sensors and array elements (3,5, 6). Recent advances in the understanding of their optical properties(10, 11, 23) offer the possibility of using such materials as solutionphase sensors (7, 8) that respond to analyte adsorption by modulation ofoptical properties, e.g., modulation of fluorescence emission. Suchsensors could be implanted, for example, into human tissue (9) toprovide real-time information about biochemical concentrationsnon-invasively.

Carbon nanotubes fluoresce in the near infrared (10, 11) and certaintypes (e.g., single walled carbon nanotubes (SWNTs) fluoresce from 900to 1600 nm) do so in a region where human tissue and fluids, e.g., wholeblood, (12) are particularly transparent to emission due to greaterpenetration and reduced auto-fluorescent background. Furthermore, SWNTshave particular advantage as sensing elements because all atoms of thenanotube are surface atoms making the nanotube especially sensitive tosurface adsorption events. However, the ability to design sensors fromcarbon nanotubes is limited by fundamental limitations in our currentability to simultaneously control the electronic, chemical and colloidalproperties of nanoparticle systems. Addition information on theproperties of carbon nanotubes is found in the art (52-57.)

For use in selective optical sensor applications for the detection ofanalytes, carbon nanotubes must retain their ability to luminesce, theymust be capable of interacting selectively with the analyte to bedetected, and the selective interaction with the analyte must affectcarbon nanotube luminescence. Nanotubes in electrical contact with eachother do not luminesce because the excited state is depopulatednon-irradiatively through inter-tube energy transfer (10). However, vander Waals forces provide large thermodynamic driving forces foraggregation of carbon nanotubes. For nanotubes to luminesce, they mustbe colloidally stabilized (to minimize or avoid aggregation). Individualfluorescent carbon nanotubes have been suspended after high energyultrasonication using charged surfactants (10, 11, 13), non-ionicpolymers (10, 22), and certain DNA sequences (14, 15). However, theseinterfaces interfere with the adsorption of charged reagents (17, 15)either via columbic interactions, or steric repulsion.

PCT published application WO03/050332 relates to the preparation ofstable carbon nanotube dispersions in liquids. PCT published applicationWO02/095099 relates to noncovalent sidewall functionalization of carbonnanotubes.

PCT published application WO02/16257 relates to polymer wrapped singlewall carbon nanotubes.

PCT published application WO03/102020 reports a method for obtainingpeptides which bind to carbon nanotubes and other carbon nanostructures.Libraries containing peptides, typically a random mixture of peptides,are selected for their binding affinity for carbon nanotubes. Details ofthe method are given. A number of peptides of specific peptide sequencewere identified as having binding affinity for carbon nanostructures,including carbon nanotubes. The sequences of a number of such peptides,particularly a set of peptides having 12 amino acids, were provided inthe published application.

In addition, dispersed nanotubes exhibit more prominent resonant Ramanscatter which is more sensitive to the environment of the nanotube (17),which may be useful in sensing applications.

Functionality must be associated with the carbon nanotube to provide forselective interaction with analytes. The inherent selectivities ofbiological molecules might, for example, be employed to provide forselective interaction of carbon nanotubes with analytes. However, toremain useful for sensing applications, nanotube functionalization mustnot disrupt nanotube optical properties (5). While it is possible tochemically attach functional groups to singly dispersed nanotubes (16),covalent functionalization of carbon nanotubes necessarily disrupts the1-D electronic structure and desired optical properties (5, 16, 23, 24).Functionalization chemistries necessarily result in a rupturing of theconjugated π-cloud along the nanotube, disrupt its optical transitionsand destroy fluorescence. Non-covalent modification using electroactivespecies, although difficult to control (17), provide a means of bothpreserving the carbon nanotube electronic structure (since no bonds arebroken) and creating sites for selective binding.

The current state of carbon nanotube chemistry is therefore paradoxical:one must chemically modify the nanotube to impart desired functionalityand selectivity toward analytes, but in doing so the 1-D electronicstructure is disrupted destroying the ability to detect analyteinteraction. Additionally, an encapsulating phase (e.g., surfactant)must be used to isolate the nanotube for colloidal stability andretention of fluorescence, and yet the stabilized nanotube must beaccessible to facilitating molecular recognition.

The present invention provides solutions to the limitations discussedabove and provides optical sensors and methods for the selectivedetection of analytes employing carbon nanotubes, particularly SWNTs.

SUMMARY OF THE INVENTION

This invention provides compositions comprising carbon nanotubes inwhich the carbon nanotubes retain their ability to luminesce and inwhich that luminescence is rendered selectively sensitive to thepresence of an analyte. More specifically, the invention providescompositions comprising individually dispersed carbon nanotubes, whichare electronically isolated from other carbon nanotubes, yet which areassociated with chemical selective species, such as polymers,particularly biological polymers, for example proteins, which caninteract selectively with, or more specifically selectivity bind to, ananalyte. Chemically selective species bind, preferably non-covalently,to the carbon nanotube and function to provide for analyte selectivity.Chemically selective species include polymers to which one or morechemically selective groups are covalently attached.

Chemically selective species also include polymers which are inherentlychemically selective in that they selectively bind to or selectivelyreact with an analyte. Analyte is detected and/or the amount present ismeasured by changes in nanotube luminescence. Chemically selectivespecies useful for analyte detection further include polymers whichinherently or via one or more covalently attached chemically selectivegroups competitively interact with (e.g., bind to or react with) anotherchemical species (for convenience herein called a sensing partner) withwhich the analyte interacts. For example, the polymer or the attachedchemically selective species can compete with the analyte for binding toa binding partner. In another example, the polymer or the attachedchemically selective species can compete with the analyte reaction withthe sensing partner (e.g., an enzyme). Analyte is detected and/or theamount present is measured by following changes in nanotube luminescenceas analyte competes with the chemically selective species with respectto interaction with the sensing partner.

The invention provides analyte sensing compositions comprisingindividually dispersed carbon nanotubes complexed with one or morechemically selective species. Chemically selective species, can provide,at least in part, for individual dispersion of the carbon nanotubes. Theanalyte sensing compositions may further comprise additional polymericspecies (including proteins (including polypeptides) polysaccharides ornon-biological polymers (such as polymeric non-ionic detergents) whichfunction primarily for facilitating individual dispersion of the carbonnanotubes and are not chemically selective species. For use incompetitive assays for detection of analyte, analyte sensingcompositions may further comprise one or more sensing partners for theanalyte.

The interaction of individually dispersed carbon nanotubes with thechemically selective species functions to couple the chemicallyselective species directly, or indirectly via one or more redoxmediators, to the electronic band structure of the nanotube. Thiscoupling allows a specific interaction of the chemically selectivespecies with the analyte to modulate the optical properties of thenanotube. More specifically, individually dispersed semiconductingsingle walled carbon nanotubes exhibit band gap fluorescence which isphoto-induced by irradiation with electromagnetic radiation ofappropriate wavelength. A specific interaction of the chemicallyselective species with the analyte can, for example, modulates nanotubeband gap fluorescence affecting fluorescence intensity, e.g., throughcharge transfer, or shifting the emission wavelength(s), which can bemediated through induced dipole or bathochromic interactions.Interaction of the analyte with the chemically selective species may forexample, increase fluorescence intensity, or decrease fluorescenceintensity or the interaction may shift one or more wavelengths offluorescence.

In analyte sensing compositions of this invention, the opticalproperties of carbon nanotubes are responsive to specific and/orselective interactions, such as binding events (including binding anddisruption of binding) or reactions, of the chemically selective specieswith an analyte. In specific embodiments, the chemically selectivespecies of this invention are biological molecules, particularlyproteins, and more specifically proteins that selectively bind to ananalyte (e.g., antibodies or antibody fragments) and even morespecifically enzymes (e.g., glucose oxidase) which bind to and catalyzea reaction of the analyte. In other specific embodiments, the biologicalmolecule may also be polysaccharides (e.g., a dextran, composed ofglucose units) which competitively binds to a binding partner of asaccharide analyte (e.g., glucose). In other specific embodiments, thechemically selective species are polymers, which are not necessarilythemselves biological molecules, but which are functionalized to containchemically selective groups or moieties which are biological molecules(e.g, biotin, ligands for biological receptors, enzyme substrates,etc.).

In specific embodiments, the carbon nanotubes exhibit band gapfluorescence, particularly near-IR fluorescence. In specificembodiments, the carbon nanotubes are single-walled, semiconductingcarbon nanotubes. In specific embodiments, the chemically selectivespecies is non-covalently complexed with the carbon nanotube. Inspecific embodiments, the chemically selective species are enzymes whichoxidize the analyte and in which this selective reaction is coupled tothe electronic band structure of the carbon nanotube via one or moreredox mediators. In specific embodiments for the detection of glucose,the chemically selective species is glucose oxidase and the redoxmediator is an electroactive species such as ferricyanide. In otherspecific embodiments for detection of glucose, the chemically selectivespecies is a dextran (or other polysaccharide that exhibits binding toglucose binding partners) and the sensing partner is a protein thatbinds to glucose (and dextran) such as concanavalin A (Con A) orapo-glucose oxidase (an inactive form of glucose oxidase which retainsbinding affinity for glucose).

In a specific embodiment, the invention provides an analyte sensingcomposition which comprises a semiconducting single-walled carbonnanotube (SWNT) complexed with one or more proteins or one or morepolysaccharides (such as a dextran) and dispersed in a liquid or solidphase wherein the SWNT/protein complex or the SWNT/polysaccharidecomplex exhibits a luminescence response, (e.g., band gap fluorescence,particularly near-IR fluorescence) on excitation with electromagneticradiation of appropriate wavelength; and wherein at least one of theproteins or one of the polysaccharides complexed with the semiconductingSWNT, which is designated the sensing protein or sensing polysaccharide,respectively, is selected such that the analyte selectively binds to orselectively reacts with the sensing protein or sensing polysaccharide.The selective interaction of the analyte and the sensing protein orsensing polysaccharide directly or indirectly modulates the opticalproperties of the SWNT/protein or polysaccharide complex, for example,the luminescence response of the SWNT/protein or polysaccharide complex.In specific embodiments, the analyte sensing composition comprises asemiconducting SWNT which is complexed to one or more polymers whereinat least one of the polymers is a chemically selective species.

In specific embodiments, sensing compositions of this invention consistessentially of a semiconducting single-walled carbon nanotube (SWNT)complexed with one or more proteins, one or more polysaccharides or oneor more non-biological polymers which is functionalized with one or morechemically selective groups (e.g., biological groups or moieties)wherein the SWNT/polymer complex exhibits band gap fluorescence andwherein at least one of the polymers complexed with the semiconductingis a sensing polymer which selectively binds to or selectively reactswith the analyte and wherein the selective interaction of the analyteand the sensing polymer directly or indirectly modulates the band gapfluorescence response of the SWNT/polymer complex. In specificembodiments, the polymer is one or more proteins or a mixture of one ormore proteins with one or more polysaccharide and or one or morenon-biological polymers and one or more of the proteins is a sensingprotein.

The sensing protein can, in specific embodiments, be an enzyme withwhich the analyte selectively reacts. In specific embodiments, theenzyme catalyzes an oxidation or a reduction of the analyte. In aspecific embodiment, the enzyme generates hydrogen peroxide on reactionwith the analyte. In more specific embodiments, the enzyme is an oxidaseand the analyte is a substrate for the oxidase. In other specificembodiments, the enzyme (where the analyte is a substrate of the listedenzyme) is selected from the group consisting of a glucose oxidase, aglucose dehydrogenase, a galactose oxidase, a glutamate oxidase, anL-amino acid oxidase, a D-amino acid oxidase, a cholesterol oxidase, acholesterol esterase, a choline oxidase, a lipoxigenase, a lipoproteinlipase, a glycerol kinase, a glycerol-3-phosphate oxidase, a lactateoxidase, a lactate dehydrogenase, a pyruvate oxidase, an alcoholoxidase, a bilirubin oxidase, a sarcosine oxidase, a uricase (alsocalled a urate oxidase), and an xanthine oxidase. Embodiments in whichthe sensing protein is an enzyme can be used in various enzyme assaysfor detection, quantitation or both of analyte, for example, incompetitive inhibition assays.

Further the analyte sensing compositions of this invention can beemployed to detect analytes that are inhibitors of enzyme activity. Inthis case, the sensing protein is an enzyme the activity of which isinhibited by the analyte. Analyte sensing compositions for the detectionand/or quantitation of an enzyme inhibitor would also comprise an enzymesubstrate, present in an amount sufficient to not be limiting to theenzyme reaction. The analyte sensing compositions of this invention canthus be employed to screen for the presence of enzyme inhibitors.

In specific embodiments, the sensing composition also contains a redoxmediator in addition to the sensing protein which may be reversibly orirreversible complexed with the semiconducting SWNT. The mediatorfunctions to alter surface charge of the carbon nanotube to modulate theluminescence response of the semiconducting SWNT/protein complex. Mostgenerally, the redox mediator is an electroactive species. In specificembodiments, the redox mediator is an electron acceptor. In specificembodiments, the mediator is a transition metal complex ion. In specificembodiments, the mediator is an electroactive species that is reduced oroxidized by reaction with a product of the reaction of the analyte witha sensing protein. In specific embodiments the mediator is an oxidizedspecies that is reduced by reaction with hydrogen peroxide generated onreaction of the analyte with a sensing protein enzyme. In a morespecific embodiment, the mediator is ferricyanate ion or ferrocyanateion.

In specific embodiments, the analyte is a natural substrate of theenzyme employed as the sensing protein. In specific embodiments, theanalyte is not the natural substrate of the enzyme, which can includechemical derivatives of the natural substrate for the enzyme. Inspecific embodiments, the enzyme is obtained from commercial sources, isisolated from natural sources, or is prepared by recombinant methods. Inspecific embodiments, the enzyme is a variant, derivative, orsemi-synthetic analog of a naturally-occurring enzyme which, forexample, has been modified by modification of one or more amino acids toexhibit altered activity, e.g., enhanced activity, compared to thenaturally-occurring enzyme, is a deglycosylated form of anaturally-occurring enzyme or a variant or derivative thereof, is formedby reconstitution of an apo-enzyme with its required co-factor (e.g.,FAD), is formed by reconstitution of an apo-enzyme with a derivatizedco-factor. Enzyme variants, derivatives or semi-synthetic analogs ofnaturally-occurring enzymes may exhibit altered substrate specificityand/or altered enzyme kinetics compared to naturally-occurring forms ofthe enzyme.

In other specific embodiments, the sensing protein is a receptor orother binding partner to which the analyte selectively binds. In thesecases, the analyte may itself be a ligand or binding partner whichselectively binds to the sensing protein, or the analyte may becovalently linked to or non-covalently complexed with a ligand orbinding partner which selectively binds to the sensing protein.Alternatively, the analyte may be covalently linked to or non-covalentlycomplexed with another chemical species to together form a ligand orbinding partner that selectively binds to the sensing protein. Thesensing protein can, for example, be an antibody or antibody fragment.

In other specific embodiments, the sensing polysaccharide is a bindingpartner to which the analyte selectively binds. Alternatively, thesensing polysaccharide binds to a binding partner to which the analytealso binds. In these cases, the analyte may be a protein or otherpolypeptide, a ligand or binding partner which selectively binds to thesensing polysaccharide, or the analyte may be covalently linked to ornon-covalently complexed with a protein or other polypeptide, ligand orbinding partner which selectively binds to the sensing polysaccharide.Alternatively, the analyte may be covalently linked to or non-covalentlycomplexed with another chemical species which together forms a ligand orbinding partner that selectively binds to the sensing polysaccharide.The sensing polysaccharide can, for example, be a polymer of amonosaccharide-analyte (e.g., a dextran for glucose detection.)

Further the analyte sensing compositions of this invention can beemployed to detect analytes that inhibit or otherwise interfere withbinding of the members of a binding pair (e.g., a ligand and itsreceptor). In this case, the sensing polymer (e.g., the sensing proteinor sensing polysaccharide) is a polymer which functions as one member ofthe binding pair, the binding of which is inhibited by the analyte.Analyte sensing compositions for the detection and/or quantitation of aninhibitor of binding would also comprise the other member of the bindingpair, present in an amount sufficient to not be limiting to binding(e.g., in the case of sensing protein which is a receptor, the othermember of the binding pair is the ligand of that receptor). The analytesensing compositions of this invention can thus be employed to screenfor the presence of inhibitors of binding of a binding pair or bindingpartners. For example, the analyte sensing compositions of thisinvention in which the sensing protein is avidin could be employed todetect the presence of inhibitors, for example competitive bindinginhibitors, of the binding of avidin to biotin.

In other specific embodiments, the sensing protein is an antibody or anantibody fragment which binds selectively or specifically to aparticular antigen and the analyte is the antigen which binds directlyto the antibody or antibody fragment or the analyte may be covalentlylinked to or non-covalently complexed with an antigen which selectivelybinds to the sensing protein. Alternatively, the analyte may becovalently linked to or non-covalently complexed with another chemicalspecies which together form an antigen that selectively binds to thesensing protein antibody or antibody fragment. The antibodies andantibody fragments employed as sensing proteins may be obtained fromcommercial sources, isolated from natural sources, or obtained byrecombinant methods.

In another specific embodiment, the analyte is an antibody or antibodyfragment and the sensing protein is an antigen of the antibody orantibody fragment, containing an epitope which selectively binds to theantibody or antibody fragment, or the sensing protein is derivatized todisplay one or more antigens which selectively bind to the antibody orantibody fragment. More specifically, the sensing protein is covalentlyderivatized, prior to complexation with the carbon nanotube, to displayone or more antigens which selectively bind to the antibody or antibodyfragment. The presence and/or concentration of the antibody or antibodyfragment in contact with the sensing composition is determined bydetection of modulation in the optical properties of the carbon nanotube(e.g., fluorescence emission) as antibody or antibody fragmentscontacting the sensing solution bind to the antigens or epitopes of thesensing protein.

In specific embodiments, the analyte is a natural ligand of a receptoremployed as the sensing protein. In specific embodiments, the analyte isnot the natural ligand of the receptor, which may include chemicalderivatives of the natural ligand, but nevertheless selectively binds tothe receptor in specific embodiments, the receptor is obtained fromcommercial sources, is isolated from natural sources, or is prepared byrecombinant methods. In specific embodiments, the receptor is a variant,derivative, or semi-synthetic analog of a naturally-occurring receptorwhich, for example, has been modified by modification of one or moreamino acids to exhibit enhanced affinity or altered affinity for aselected ligand, is a deglycosylated form of a naturally-occurringreceptor or a variant or derivative thereof, which retains function as areceptor for binding selectively to a ligand.

In another aspect, the invention provides materials and methods fordetecting analytes by competitive binding assays. Most generally, inthis embodiment, the analyte is a ligand or antigen to which a bindingpartner (e.g., a ligand receptor or antibody or antibody fragment)selectively binds. The analyte may also be a substrate which bindsselectively or specifically to an enzyme. In one embodiment, the sensingcomposition for the competitive binding assay comprises carbon nanotubesnon-covalently complexed with a sensing polymer, such as a protein or anon-ionic detergent, which is covalently derivatized, prior tocomplexation with the carbon nanotube, to display one or more ligands orantigens which selectively bind to the binding partner. In thisembodiment the sensing composition further comprises the binding partnerwhich is, at least in part, bound to the one or more ligands or antigenscovalently displayed on the sensing polymer. Free analyte coming intocontact with the sensing composition, at a sufficient concentration,binds to the binding partner in the sensing composition, displacing, atleast in part, binding partners, that were bound to the ligand orantigens covalently displayed on the sensing polymer. Displacement ofthe binding partner from the carbon nanotube/sensing polymer complexmodulates the optical properties (e.g., fluorescence emission) of thecarbon nanotube. Detection and analysis of the modulation allowsdetection of the analyte and/or measurement of the concentration ofanalyte in contact with the sensing composition. Standard methods ofanalysis used in competitive binding assays can be employed to determineanalyte concentrations. The ligand or antigen covalently bound to thesensing polymer may be chemically identical to the analyte to bedetected except that the ligand or antigen is covalently attached to thesensing polymer. Alternatively, the ligand or antigen covalently boundto the sensing polymer can be a chemical variant of the analyte (e.g., apolymer of the analyte or containing the analyte) which neverthelessbinds to the binding partner. The analyte variant covalently bound tothe sensing polymer can be selected to have binding affinity for thebinding partner that is the same as or different from that of theanalyte for the binding partner.

In a specific embodiment, the sensing polymer is a protein. In aspecific embodiment, the sensing polymer is a polysaccharide. In aspecific embodiment, the sensing polymer is an organic polymer that isnot a protein, a nucleic acid or a carbohydrate. In another embodimentthe sensing polymer is poly(ethylene glycol). In a specific embodimentuseful in a competitive binding assay, the sensing polymer ispoly(ethylene glycol) to which one or more analytes or analyte variantsare covalently attached. In a more specific embodiment, for use indetection of a selected steroid, the sensing polymer is poly(ethyleneglycol) to which one or more of the selected steroids or steroidvariants are covalently attached. In a more specific embodiment, for usein detection of 17β-estradiol, the sensing polymer is poly(ethyleneglycol) to which one or more 17β-estradiols or one or more 17β-estradiolvariants are covalently attached. In another embodiment the sensingpolymer is a polyoxyethylene sorbitan fatty acid ester, such as amonolaurate ester (such as Tween 20™) For use in this invention as asensing polymer, polyoxyethylene sorbitan fatty acid esters can befunctionalized with one, two, three or more (if functionalization sitesare available) of the same chemical species (or moiety) that isselective for an analyte of interest. In a specific embodiment useful ina competitive binding assay, the sensing polymer is a polyoxyethylenesorbitan fatty acid ester to which one or more analytes or analytevariants are covalently attached. In a more specific embodiment, for usein detection of one member of a selected binding partner pair (e.g.,avidin, strepavidin), the sensing polymer is a polyoxyethylene sorbitanfatty acid ester to which the other member of that binding partner pair(e.g., biotin) is covalently attached.

The semiconducting SWNT/sensing polymer complex is present in theanalyte sensing composition in an amount sufficient to generate aluminescence response of sufficient intensity such that a modulation inthat response resulting from the interaction of the analyte with thesensing polymer is detectible. Preferably, the semiconductingSWNT/sensing polymer complex is provided in an amount sufficient toallow detection of the analyte at a selected lower concentration limit.Preferably the analyte sensing composition does not contain anysubstantial amounts of carbon nanotubes which are not complexed withsensing polymer. Preferably, the analyte sensing composition does notcontain any substantial amount of free sensing polymer that is notcomplexed with a carbon nanotube or other carbon nanostructuredcomponent of the sensing solution.

The analyte sensing solution may contain other functional components asneeded in an amount sufficient to provide the desired sensingfunctionality. For example, the sensing solution may contain enzymeco-factors, co-reactants, oxidation agents or reducing agents as may beneeded to facilitate selective reaction of the analyte mediated by thesensing protein enzyme. The analyte sensing solution may containadditional functional components, as needed and in an amount sufficientto provide the desired functionality, which facilitate ligand-receptorbinding or antigen-antibody (or antibody fragment) binding.

The analyte sensing composition may comprise carbon nanotubes complexedwith one or more chemically selective species which are dispersed in asolid or semi-solid matrix wherein the complexed carbon nanotubesexhibit luminescence and the solid or semi-solid matrix is selectivelypermeable to the analyte.

The invention also provides a sensor element for detecting an analytewhich comprises:

-   -   a selectively porous container for receiving and retaining the        components of a sensing composition, and    -   an analyte sensing composition, as described above, within the        selectively porous container wherein the selectively porous        container is sufficiently porous to allow the analyte to enter        the container without allowing the functional components of the        sensing solution to exit the container.

The sensor element can alternatively comprise an analyte sensingcomposition dispersed in a solid or semi-solid matrix wherein the solidor semi-solid matrix is selectively porous to the analyte.

The sensor element of the invention may be a tissue implantable sensorelement. Tissue implantable sensor elements can be prepared employingbiocompatible materials. Tissue implantable sensor elements can beencased in a biocompatible hydrogel matrix. The hydrogel matrix cancontain one or more growth factors which induce vascularization intissue in which the sensor element is implanted.

The invention also provided a sensing system for detecting the presenceof an analyte or for determining the amount of an analyte whichcomprises:

-   -   an analyte sensing composition comprising a semiconducting        SWNT/complexed with one or more polymers including at least one        sensing polymer, as described above,    -   a source of electromagnetic radiation, e.g., light, for exciting        luminescence of the SWNT/protein complex and    -   a detector for detecting the luminescence response generated by        the SWNT/sensing polymer complex on reaction of the analyte        mediated by the sensing polymer or on binding of the analyte        (directly or indirectly) to the sensing polymer.

In specific embodiments the sensing polymer is a sensing protein or asensing polysaccharide. In specific embodiments the sensing polymer is apolymer other than a polynucleotide.

The analyte sensing solution is brought into contact with an environment(e.g., a liquid or solid sample, a biological fluid, an organism, amicroorganism or medium containing microorganisms, an animal, a mammal,a human, a cell growth medium, etc.) such that analytes that may be inthe environment can enter into and interact with the sensing compositionand the sensing polymer therein to modulate the luminescence response ofthe SWNT/sensing polymer complex.

In a specific embodiment, the sensing composition is retained in aselectively porous sensing element which can be placed in contact withan environment which may contain the analyte. The selectively poroussensing element is sufficiently porous to allow the analyte to enter thesensing element without allowing any substantial release of thefunctional components of the sensing composition. In a specificembodiment, the environment which the sensor element contacts is atissue or body fluid. In a specific embodiment, the sensing element isinserted into the body or a body part of an animal, including any mammaland including a human.

In a more specific embodiment, the analyte is glucose and the sensingpolymer is glucose oxidase. In a yet more specific embodiment, theanalyte is glucose, the sensing protein is glucose oxidase and themediator is ferricyanate. In another embodiment, the analyte is glucoseand the sensing polymer is a dextran and the sensing composition furthercomprises a sensing partner which is a glucose binding partner, andwhich more specifically is concanavalin A or apo-glucose oxidase.

In another aspect, the invention provides a method for preparing acarbon nanotube complex with a polymer which comprises the steps:

-   -   a. providing surfactant dispersed carbon nanotubes;    -   b. contacting the surfactant dispersed carbon nanotubes with the        polymer to form a mixture    -   c. dialyzing the mixture to remove the surfactant such that        carbon nanotube complexes with the polymer are formed.

In specific embodiments the polymer is a biological polymer such as aprotein or other polypeptide or a polysaccharide.

In preferred methods, the polymer is a protein, including a solubleprotein (typically soluble in aqueous media) or an amphiphilic protein,and the surfactant is non-denaturing. The surfactant is a surfactantthat can be removed by dialysis and is typically not a polymericsurfactant. The surfactant can, among others be an anionic surfactant,such as a cholate, a nonionic surfactant or a zwitterionic surfactantthat is not polymeric. In preferred embodiments, the relative amounts ofpolymer and carbon nanotube in the mixture that is to be dialyzed isselected to maximized the formation of carbon nanotube/polymer complexesand minimize the amount of free-polymer in the mixture after dialysis.Preferably the relative amount of carbon nanotube to polymer in themixture is selected to obtain a monolayer or less of the polymer(including protein) on the carbon nanotube.

The methods of this invention can also be employed when the protein orother polymer is not soluble in an aqueous medium, for example when theprotein is a membrane protein. In this case, the protein or otherpolymer may be initially dispersed with a surfactant (or mixture ofsurfactants) which does not denature the protein or otherwisedetrimentally affect the polymer, as is known in the art, the dispersedprotein or other polymer is then contacted with the dispersed carbonnanotubes and the surfactant(s) are thereafter removed from the systemto form the carbon nanotube/protein complex.

Dialysis is performed as is known in the art using surfactant-freemedium (aqueous medium) to remove surfactant without significant loss ofcarbon nanotubes or polymer, and to retain any function of the polymer.More specifically, the dialysis is performed under conditions selectedto retain the biological function of a protein to be complexed with acarbon nanotube.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C. FIG. 1A is a graph of normalized fluorescence intensity ofsemiconducting SWNTs during equilibrium dialysis of a SWNT/cholatesuspension with added glucose oxidase to form SWNT/glucose oxidasecomplexes. Fluorescence of semiconducting SWNTs is measured usingtransient fluorescent spectroscopy with 785 nm excitation. Fluorescenceintensity is normalized to the Raman tangential mode at 1592 cm⁻¹ as afunction of dialysis time. Normalized fluorescence intensity decreasesas cholate is removed. A phase change, indicated by a sharp drop innormalized fluorescence intensity, occurs at about 3.8 hrs after thestart of dialysis, where surface assembly occurs. FIG. 1B provides acomparison of the emission spectrum observed at the start of dialysis(t=0 hrs) from individual dispersed semiconducting SWNTs in aSWNT/cholate dispersion and the emission spectrum observed aftermacromolecular complex formation, here, SWNT/glucose oxidase complexformation. The emission spectrum shifts by about 10 meV and fluorescenceintensity is reduced by a factor of 2.2. This shift is believed to bethe result of increased polarity at the nanotube surface due to waterpenetration through cavities in the adsorbed enzyme layer shifts. FIG.1C. The assembly event is concerted, as measured using the emission peakcenter, and occurs over an extremely narrow range of cholateconcentrations as show in this graph of peak shift as a function oftime.

FIGS. 2A-C: Non-covalent functionalization of the nanotube surface.Illustrating the effect of ferricyanide ion at the nanotube surface.FIG. 2A. Through gaps between macromolecules on the nanotube surface,the mediator can interact directly with the nanotube surface and allowthe surface chemistry of an individual nanotube to be probed without anintervening surfactant layer. FIG. 2B. Electroactive species, such asferricyanide ion exemplified herein, can withdraw electronic densityfrom the nanotube valence bands, and remain bonded to the nanotube inthe absence of a covalent bond. Both the ferricyanide (shown) andferrocyanide ions react in this way, withdrawing electron density fromthe nanotube with increasing surface coverage. FIG. 2C. The process ofwithdrawal of electron density is tracked using SWNT fluorescence,scaled by the initial emission intensity I_(o) which trace single anddual site binding isotherms for ferricyanide and ferrocyanide ions,respectively.

FIG. 3: Schematically illustrates coupling of an enzyme reaction (thatof glucose oxidase) to the luminescence response of a semiconductingSWNT. The multi-functional tailoring on the surface of an individualcarbon nanotube with enzyme and mediator couples specific binding to theoptical response of the substrate. The immobilized enzyme provides sitesfor β-d-glucose specificity with surface functionalization of thenanotube surface with a mediator Fe(CN)₆ ⁻³. Reaction at the enzymecoverts glucose to gluconolactone, and the hydrogen peroxide co-productis detected by interaction with the mediator at the nanotube surface.The arrangement allows for the engineering of surface functionalitywhile retaining nanotube electronic structure and fluorescent emission.

FIGS. 4A-C: Illustrates a tissue implantable device for analytedetection, exemplified for glucose detection: FIG. 4A. A micro-dialysiscapillary 100 (MWCO=13 kDa) is loaded with the functionalized nanotubes(GOx-SWNT-Fe(CN)₆ ⁻³) in solution allowing glucose to diffuse throughthe dialysis membrane (101) with containment of the sensing medium. Themicro-dialysis capillary may be inserted into tissue, for example at afinger tip as illustrated. FIG. 4B is a graph which shows the effect ofinjection of 62.5 mM of ferricyanide into a suspension containingsemiconducting SWNT/GOx. A rapid diminution in the scaled near-IRfluorescence occurs due to the interaction of the ferricyanide with theSWNT. The response is normalized by the pristine I_(a) and fully reactedI_(b) emission. Subsequent addition of 1.4 mM, 2.4 mM, and 4.2 mM ofglucose, as indicated, causes a quantitative restoration of thefluorescence. The sensing medium exhibited a detection limit of 34.4 μMand response time less than 80 s. FIG. 4C is a graph of SWNTfluorescence response as a function of glucose concentration. Theresponse function relates the normalized intensity to the local glucoseconcentration.

FIG. 5 is a graph showing transient fluorescence measurements of theSWNT/cholate/GOx mixture (red) and a control dialysis sample of theSWNT/cholate suspension (blue) as a function of dialysis time.

FIG. 6 is a graph of normalized fluorescence for the [6,5]carbonnanotube as a function of the weight ratio of GOx (protein) to SWNT inSNWT/cholate/GOx samples after (20 hrs) dialysis.

FIG. 7: Ultraviolet visible near infrared absorption spectrum ofGOx/SWNT complex maintained at 37° C. (spectrum a). After addition of 48mM ferricyanide (spectrum b) E11 transitions above 1100 nm decreasewhile transitions between 900 and 1100 nm remain unchanged. The apparentincrease in absorption between 500 and 700 nm is due to ferricyanideabsorption in the visible. The solution was then titrated with 2 mMhydrogen peroxide (spectrum c) resulting in only a partial restorationof the longest wavelength excitations and no change in the otherspectral features.

FIG. 8 is a graph showing the effect of additions of ferricyanide to asensing medium containing GOx-SWNT complexes.

FIGS. 9A and 9B are graphs showing fluorescence response in controlglucose sensing experiments. FIG. 9A Fluorescence of 2% cholatesuspended SWNT on addition of glucose. FIG. 9B. Fluorescence of GOx-SWNTon addition of glucose. Mediator, ferricyanide ion, was not added toeither of these controls. The control samples were both buffered (pH7.4) and maintained at 37 C. No significant change in fluorescence wasobserved on glucose addition in these control experiments.

FIG. 10 shows normalized response of Fe(CN)₆ ³⁻ scattering intensity (□)monitored via Raman scattering at 785 nm at 2132 cm⁻¹ after addition of20 mM glucose. The glucose was added to a solution of 0.0127 mM GOx and62.5 mM Fe(CN)₆ ³⁻ in 0.5 M phosphate buffer at pH 7.4 and 37° C. Thesolid line is the fit of the calculated normalized ferricyanideconcentration.

FIG. 11 shows the change in glucose concentration after first glucoseinjection in sensing buffer calculated from fluorescent data (□) and thecorresponding model using an effectiveness parameter of 5.61 and kineticparameters K_(m)=4.21 mM and k₂=293.4 min⁻¹.

FIG. 12 is a schematic of an exemplary sensor comprising a sensorelement which is a dialysis capillary (200) based on competitive bindingof an antibody to an analyte derivative (bound to a sensing polymer inthe SWNT/sensing polymer complex) and a free analyte. The sensor isexemplified for detection of estradiol-17β (E2).

FIG. 13 is a graph showing the change in fluorescence change of Avidinsuspended SWNT with additions of Biotin, see the Examples.

FIG. 14 A is a schematic illustration of a carbon nanotube opticalsensor for glucose detection that can be implanted beneath the skinwhere the sensing medium is contained in a dialysis capillary encased ina biocompatible hydrogel matrix. FIG. 14 B is a scheme showing themechanism of action of a glucose sensor employing a SWNT/dextran complexand based on competitive binding of a protein (having binding affinityfor glucose and dextran, e.g., conA or apo-GOx). Binding of glucose tothe protein causes SWNT fluorescence attenuation.

FIG. 15 is a schematic illustration of a sensing system of thisinvention in which a sensing composition is introduced into a sensingelement (e.g., a capillary porous to analyte but not to sensingcomponents). The system has a light source for exciting luminescence ofthe carbon nanotube and a detector for detecting that luminescence andchanges in the luminescence.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates most generally to non-covalent complexes of carbonnanotubes with polymers, particularly sensing polymers and moreparticularly with proteins, polypeptides and polysaccharides.

The present invention provides sensors which comprise analyte sensingcomposition which in turn comprise complexes of carbon nanotubes withsensing polymers. In these complexes, the sensing polymer isnon-covalently complexed with the carbon nanotube. Preferably, in thesensing composition, the sensing polymer is complexed with the carbonnanotube to provide individually dispersed carbon nanotubes with noelectronic interaction or minimal electronic interaction with othercarbon nanotubes in the composition. The sensing polymer provides forselective interaction with an analyte or specific interaction with ananalyte. The term “specific” is used to indicate an interaction that canbe used to distinguish the analyte from most other chemical speciesexcept optical isomers, isotopic variants (i.e., where one or more atomsare enriched in a given isotope, e.g., deuterated species) and perhapscertain isomers. The term “selective” is used to indicate an interactionthat is sufficiently specific that it can be used to distinguish theanalyte in practice from other chemical species in the system in whichthe sensor and sensing composition is to be employed. The term“selective binding” is thus used to refer to a protein, other polymer orother chemical species that binds to a limited set of other chemicalspecies (usually species that are related in chemical structure).Enzymes, antibodies (and antibody fragments) and receptors, among otherproteins, exhibit selective binding which may in some cases be specific.Other polymers, such as polysaccharides may functions as ligands (e.g.,for binding to a protein) or as a member of a binding pair. In themethods and devices of this invention “selective binding” can providethe selectivity needed to detect a selected analyte (or relatively smallgroup of related analytes) in a complex mixture, e.g., in a biologicalfluid or tissue. For example, selective binding of a substrate to anenzyme can provide the desired level of selectivity needed to detect aselected analyte (which is the enzyme substrate). Selective bindingincludes “specific binding” which is intended to indicate more limitedbinding which can be used to distinguish a binding partner from mostother chemical species except optical isomers, isotopic variants andperhaps certain structural isomers. Sensing polymers of this inventioncan be selected to provide selective or specific interactions with oneor more analytes (preferably one analyte).

The term analyte is used generally herein to refer to any chemicalspecies which is to be detected or the quantity of which is to bedetermined. Analytes include small molecules, such as sugars, steroids,antigens and polymeric species such as proteins (e.g., enzymes,antibodies, antigens). In specific embodiments, analytes are one memberof a binding partner pair. In specific embodiments analytes aremonosaccharides. In a specific embodiment the analyte is glucose.Methods, devices and compositions herein are particularly well suited tothe detection and quantitation of analytes in solutions, such as inbiological fluids. Methods, device and compositions herein are alsoparticularly well suited to the detection and quantitation of analytesin biological tissues.

The sensing polymer may be formed by derivatization of a polymer, e.g.,poly(ethylene glycol), with one or more chemically selective specieswhich provide for selective or specific interaction with one or moreanalytes. Additional polymers that may be derivatized to form sensingpolymers include among others, poly(vinyl alcohol), poly(vinylchloride), (e.g., and copolymers thereof, polysorbitan esters (e.g.,polyoxyethylene sorbitan fatty acid esters.) Each sensing polymer may bederivatized to carry one, two or more chemically selective species ormoieties which are selective for the same analyte. A sensing polymer maybe derivatized to carry one, two or more chemically selective species ormoieties which are selective for the same or different analyte. Thus asingle, carbon nanotube/sensing polymer complex may be responsive toone, two or more analytes. In specific embodiments, a sensing polymercontains one covalently bound chemically selective species or moietywhich is selective for a single analyte of interest. The use of polymerswhich carry one such selective chemical species or moiety may bebeneficial to prevent aggregation of carbon nanotube/sensing polymercomplexes. Such aggregation may be detrimental in analyte sensingapplications. The chemically selective species or moiety may be directlybonded to the polymer or indirectly bonded through a linker group.

The sensing polymer may be a sensing protein or a sensingpolysaccharide. A sensing protein has function for selective interaction(or specific interaction) with an analyte. The sensing protein may be anaturally-occurring protein or recombinant protein that exhibits as aconsequence of its protein structure a selective interaction with ananalyte. The sensing protein can interact directly with an analyte(e.g., by binding or reaction) or can interact indirectly with theanalyte by interaction (e.g., by binding or reaction) with anotherchemical species which in turn interacts with the analyte. The sensingprotein may be formed by chemical derivatization of a protein that doesnot exhibit any selective interaction with an analyte. For example, thesensing protein may be formed from a protein that is derivatizedcovalently to carry one or more chemically selective species (ormoieties) which individually or collectively provide for selectiveinteraction with one or more analytes. Proteins may be derivatized atone or more termini or at one or more amino acid side changes (e.g.,those of lysine, glutamine, arginine, serine, aspartate, glutamate,etc.) to provide for chemical selectivity. Proteins useful as sensingproteins include those which are derivatized at one or more terminiand/or one or more amino acid side chains to carry one or more steroidsor steroid derivatives.

A sensing polysaccharide has function for selective interaction (orspecific interaction with) analyte. The sensing polysaccharide may benaturally-occurring, for example isolated from nature,chemically-derivatized, chemically-modified or chemically-synthesized.The sensing polysaccharide can interact directly with an analyte (e.g.,by binding or reaction) or can interact indirectly with the analyte byinteraction (e.g., by binding or reaction) with another chemical specieswhich in turn interacts with the analyte. The specific structure of thepolysaccharide or the presence of a specific monosaccharide mayfacilitate a selective interaction with an analyte. The sensingpolysaccharide may be formed by chemical derivatization or modificationof a polysaccharide that does not exhibit any selective interaction withan analyte. For example, the sensing polysaccharide may be formed from apolysaccharide that is derivatized covalently to carry one or morechemically selective species (or moieties) which individually orcollectively provide for selective interaction with one or moreanalytes. Polysaccharides may be derivatized at any available locationof the polymer that is reactive to provide for chemical selectivity.Polysaccharides that are useful, for example, as sensing polymersinclude those polysaccharides which bind to a binding partner, forexample a protein, that also binds to a monosaccharide analyte.Polysaccharides include those having 10 or more saccharide monomers.Polysaccharides include those having 20 or more saccharide monomers

Carbon nanotubes are carbon nanostructures in the form of tubes, rangingin general in diameter from about 0.5-200 nm, (more typically forsingle-walled carbon nanotubes from about 0.5-5 nm) The aspect ratio ofnanotube length to nanotube diameter is greater than 5, ranges from10-2000 and more typically 10-100. Carbon nanotubes may be single-wallednanotubes (a single tube) or multi-walled comprising with one or moresmaller diameter tubes within larger diameter tubes. Carbon nanotubesare available from various sources, including commercial sources, orsynthesis employing, among others, arc discharge, laser vaporization,the high pressure carbon monoxide processes.

The following references provide exemplary methods for synthesis ofcarbon nanotubes: U.S. Pat. No. 6,183,714; PCT/US99/25702;PCT/US99?21367; A. Thess et al. Science (1996) 273:483; C. Journet etal. Nature (1997) 388, 756; P. Nikolaev et al. Chem. Phys. Lett. (1999)313:91; J. Kong et al. Chem. Phys. Lett. (1998) 292: 567; J. Kong et al.Nature (1998) 395:878; A. Cassell et al. J. Phys. Chem. (1999) 103:6484;H. Dai et al. J. Phys. Chem. (1999) 103:11246; Bronikowski, M. J., etal., Gas-phase production of carbon single-walled nanotubes from carbonmonoxide via the HiPco process: a parametric study. J. Vac. Sci. Tech.A, 2001. 19(4): p. 1800-1804; Y. Li et al. (2001) Chem. Mater. 13:1008;N. Franklin and H. Dai (2000) Adv. Mater. (2000) 12:890; A. Cassell etal. J. Am. Chem. Soc. (1999) 121:7975; International Patent ApplicationWO 00/26138. International Patent Applications WO 03/084869 and WO02/16257 also provide an overview of synthetic methods for thepreparation of single-walled carbon nanotubes as well as methods forpurification of carbon nanotubes and the removal of catalyst andamorphous carbon.

Carbon nanotubes produced in such methods are typically poly-dispersesamples containing metallic and semi-conducting types, withcharacteristic distributions of diameters [28].

A method for separating single-walled carbon nanotubes by diameter andconformation based on electronic and optical properties has beenreported (Smalley et al. International Patent Application WO 03/084869.The method can be employed to prepare carbon nanotube preparationshaving enhanced amounts of certain single walled carbon nanotube types.Narrow (m, m)-distributions of single-walled carbon nanotubes arereported using a silica-supported Co—Mo catalyst [30]. M. Zheng et al.Science (2003) 302 (November) 1545 report nanotube separation by anionexchange chromatography of carbon nanotubes wrapped with single-strandedDNA. Early fractions are reported to be enriched in smaller diameter andmetallic nanotubes, while later fractions are enriched in largerdiameter and semi-conducting nanotubes.

Carbon nanotube compositions generally useful in sensors of thisinvention are those which exhibit optical properties which are sensitiveto the environment of the nanotube, i.e., optical properties which canbe modulated by changes in the environment of the nanotube. Morespecifically, carbon nanotube compositions useful in sensors of thisinvention comprise semiconducting SWNTs which can exhibit luminescence,and more specifically which exhibit photo-induced band gap fluorescence.Carbon nanotube compositions which exhibit luminescence comprise SWNTswhich when electronically isolated from other carbon nanotubes exhibitluminescence, including fluorescence and particularly those whichexhibit fluorescence in the near-IR. Carbon nanotube compositions ofthis invention comprise individually dispersed semiconducting SWNTsexhibiting luminescence, particularly photo-induced band gapfluorescence. Carbon nanotube compositions of this invention may alsoinclude MWNT and other carbon nanomaterials as well as amorphous carbon.Preferably carbon nanotube compositions of the invention comprise asubstantial amount of semiconducting SWNTs, e.g., 25% or more by weightof such SWNTs. More preferably the carbon nanotube compositions of thisinvention comprise a predominance of semiconducting SWNTs (i.e., 50% ormore by weight of semiconducting SWNTs). In general, carbon nanotubecompositions will contain a mixture of semiconducting SWNTs of differentsizes which exhibit fluorescence at different wavelengths.

Single walled carbon nanotubes are sheets of graphene—single layer ofgraphite—rolled into a molecular cylinder [25] and indexed by a vectorconnecting two points on the hexagonal lattice that conceptually formsthe tubule with a given “chiral” twist [1, 2, 28]. Hence, (n,m)nanotubes are those formed by connecting the hexagon (as shown in thecited references) with one n units across and m units down (n>m byconvention.) Carbon nanotubes have a fascinating relationship betweengeometric and electronic structure: the 1-D nature of the nanotubeexerts a unique quantization the circumferential wave-vector and hence,simple perturbations of this chirality vector yield enormous changes inmolecular properties [25], [26]. When |n−m|=0, the system is trulymetallic in nature while if |n−m|=3q (with q a nonzero integer) thenanotube possesses a small curvature induced gap and if |n−m|≠3q thenthe system is semiconducting with a measurable band-gap.

The sensing composition optionally contains SWNTs that are notsemiconducting, i.e. metallic SWNTs, that are complexed with one or moreproteins or other polymers, SWNTs (semiconducting or metallic) that arefully or partially complexed with proteins and/or polymers and/orsurfactants, other carbon nanotubes or other carbon nanostructuredmaterials that are complexed with protein (which may or may not besensing proteins), polymers (which may or may not be sensing polymer)and/or surfactant, as well as aggregates, including ropes, of SWNTs, oraggregates of other carbon nanotubes or nanostrutured materials. Thesensing composition may further contain amorphous carbon and otherbyproducts of carbon nanotube synthesis, such as residual catalyst.Preferably, the types and levels of any of these optional components issufficiently low to minimize detrimental affect on the function of thesensing solution.

An electroactive species most generally is a species, e.g., a molecule,complex or polymer, that can function for transport of electrons orholes, e.g., can function for electron transfer.

A redox mediator is an electron transfer agent which functions forcarrying electrons between a chemical species and an electrode orbetween two chemical species. More specifically a redox mediator is asubstance (or substances) that facilitates the flow of electrons in areduction-oxidation reaction. In certain methods and devices of thisinvention the redox mediator functions to carry electrons from ananalyte or a reaction product of an analyte to a carbon nanotube.Electron transport between the carbon nanotube and the analyte or itsreaction product modulates the optical properties of the carbon nanotubewhich can be employed to detect analyte or the generation of reactionproducts from the analyte.

In general any organic or organometallic redox species can be used as aredox mediator in this invention. More than one redox mediator may beinvolved in the electron transport from the analyte to the carbonnanotube. Redox mediators include among others transition metalcompounds or complexes, for example, compounds or complexes of osmium,ruthenium, iron, iridium, vanadium, and cobalt. Redox mediators, morespecifically include, metal complexes, particularly transition metalcomplexes, particularly metal complexes of osmium, ruthenium, iron,iridium, vanadium, copper, aluminum and cobalt, having one or moreligands which are halogens (e.g., Cl), OH, groups, CN groups (cyanogroups), N-containing heterocycles (e.g., pyridine and/or imidazole orderivatives thereof, metalocenes or derivatives thereof, includingferrocene, nickelocene, etc. and derivatives thereof (e.g.,dimethylferrocene, decamethylferrocene, etc.)). Redox mediators includeamong others ferricyanide, ferrocyanide, Cr(OH)₃, Al(III)(OH)₃, andCu(II)₂Fe(II)(CN)₆. Organic redox species useful as redox mediatorsinclude among others, organic dyes (e.g., viologens or substitutedviologens (e.g., methylviologen), phthalocyanines, quinones (e.g.,naphthoquinone, phenoquinone, benzoquinone, naphthenequinone, and thelike including derivatives thereof, electroactive polymers (such aspolypyroles or derivatized polypyroles), tetrathiafulvalene (TTF) andderivatives thereof, dopamine and derivative thereof, epinephrine andnorepinephrine and derivatives thereof. tetracyanoquinodimethane (TCNQ)and derivatives thereof, phenazine methosulfate or phenazine ethosulfateand derivatives thereof. Exemplary redox mediators for use with specificenzymes are provided in U.S. Pat. No. 5,413,690, which is incorporatedby reference herein, at least in part, for a description of such redoxmediators.

Carbon nanotube/polymer complexes of this invention can be made byinitial formation of individually dispersed carbon nanotubes.Individually dispersed nanotubes are formed essentially as previouslydescribed (10) by dispersion of carbon nanotube product in aqueoussurfactant solution employing high-sheer mixing and sonication todisperse the nanotubes in surfactant, followed by centrifugation toaggregate bundles or ropes of nanotubes and decanting of the upper75-80% of supernatant to obtain micelle-suspended carbon nanotubesolutions or dispersions (20-25 mg/L). Surfactant-dispersed carbonnanotubes are contacted with polymer solutions, preferably aqueoussolutions of polymer, and subjected to dialysis under conditions inwhich the surfactant is removed without removal of the polymer or carbonnanotube. As surfactant is removed by dialysis, carbon nanotube/polymercomplexes are formed.

The amount and type of surfactant employed for dispersion of carbonnanotubes can be readily determined employing methods that arewell-known in the art. As noted in detail below, the surfactant employedmust be compatible with the components of the sensing compositions,particularly with the sensing polymer, specifically with the sensingprotein. The surfactant must not destroy the function of the sensingpolymer or sensing protein. In certain cases, the surfactant must be anon-denaturing surfactant that does not significantly detrimentallyaffect the function (e.g., binding or enzymatic function) of the proteinor other polymer. The amount of surfactant needed to disperse the carbonnanotubes can be determined by routine experimentation. It is preferredto employ the minimum amount of surfactant needed to provideindividually dispersed carbon nanotubes. Surfactants are typicallyemployed between about 0.1% to about 10% by weight. (more typically from0.5% to 5% by weight) in aqueous solution to disperse carbon nanotubes.

For the formation of carbon nanotube/protein complexes, the surfactantoriginally employed (1% by weight in water) sodium dodecylsulfate (SDS)to form the individually dispersed carbon nanotubes is replaced with anon-denaturing surfactant, e.g., sodium cholate (2% by weight in water).Surfactant-dispersed carbon nanotubes are contacted in aqueous solutionwith functional protein or other polymer and subjected to dialysis underconditions in which the surfactant is removed without removal of theprotein or carbon nanotube and the protein retains function. Assurfactant is removed by dialysis, carbon nanotube/protein complexes areformed. The surfactant employed is of sufficiently low molecular weightto be removed by dialysis while the polymer is not.

Complexes of carbon nanotubes with chemically selective polymers(sensing polymers) can be prepared by methods other than the dialysismethod specifically described herein. In some cases, the polymer may becomplexed with the nanotube simply by contacting the nanotube with asufficient amount of polymer and apply vigourous mixing (e.g.,sonication), if necessary to obtain dispersed nanotubes. In other cases,an already dispersed nanotube composition comprising surfactant orpolymer which functions for dispersion of the nanotube may be contactedwith a sufficient amount of the sensing polymer and if necessary applyvigorous mixing to displace at least a portion of the surfactant orpolymer already associated with the nanotube.

The preparation of surfactant dispersed carbon nanotubes employsvigorous mixing, for example high shear mixing, which may be providedusing a high speed mixer, a homogenizer, a microfluidizer or otheranalogous mixing methods known in the art. Sonication, including variousultrasonication methods can be employed for dispersion. Preferredmethods for dispersion involve a combination of high sheer mixing andsonication.

International Patent Application WO 03/050332 reports the preparation ofstable carbon nanotube dispersions in liquids. International PatentApplication WO 02/095099 reports noncovalent sidewall functionalizationof carbon nanotubes.

In specific embodiments, analyte sensing compositions of this inventioncomprise one or more carbon nanotube/protein complexes. In thesecomplexes, one or more protein molecules are non-covalently associatedwith the carbon nanotube. Preferably, the protein molecule or moleculescomplexed with the carbon nanotube provide monolayer coverage or less ofthe carbon nanotube by protein.

In preferred carbon nanotube/protein complexes of this invention, thecomplexed protein retains its biological function and the complexedcarbon nanotube is a semi-conducting carbon nanotube which exhibits bandgap fluorescence.

In specific embodiments, analyte sensing compositions of this inventioncomprise one or more carbon nanotube/polysaccharide complexes. In thesecomplexes, one or more polysaccharide molecules are non-covalentlyassociated with the carbon nanotube. Preferably, the polysaccharidemolecule or molecules complexed with the carbon nanotube providemonolayer coverage or less of the carbon nanotube by protein.

In preferred carbon nanotube/polysaccharide complexes of this invention,the complexed polysaccharide retains its biological function and thecomplexed carbon nanotube is a semi-conducting carbon nanotube whichexhibits band gap fluorescence.

Surfactants preferred for use in the methods herein are non-denaturingand can be removed by dialysis (i.e., are dialyzable). Non-denaturingsurfactants include anionic surfactants, non-ionic surfactants andzwitterionic (or amphoteric) surfactants. The term denature (ordenaturing) is used herein with respect to protein structure andfunction. A denatured protein has lost its functional structure. Contactwith surfactants, as well as other environmental changes (e.g.,temperature or pH changes), can cause structural changes in proteins,and these structural changes can affect one or more of the biologicalfunctions of the protein. For example, a denatured enzyme will no longerexhibit enzymatic function. Contact with a non-denaturing surfactantdoes not have any significant detrimental affect on one or more of thebiological functions of a given protein. Denaturing can affect enzymaticactivity, protein binding interactions and other biological functions ofa protein. A normally denaturing surfactant may function as anon-denaturing surfactant over a selected concentration range or withrespect to certain proteins which are more resistant to its denaturingeffect than most other proteins.

Non-denaturing surfactants include, among others, bile acids andderivatives of bile acids, e.g., cholate (salts of cholic acid,particularly sodium cholate), deoxycholate (salts of deoxycholic acid,particularly sodium deoxycholate), sulfobetaine derivatives of cholicacid, particularly3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS);carbohydrate-based surfactants, for example, alkyl glucosides,particularly n-alkyl-β-glucosides (more specifically,n-octyl-α-glucoside (OG)), alkyl thioglucosides, particularlyn-alkyl-β-thioglucosides (more specifically, n-octyl-β-thioglucoside(OTG)); alkyl maltosides, particularly n-alkyl-β-maltosides (morespecifically, n-dodecyl-β-glucoside); alkyl dimethyl amine oxides (e.g.,(C6-C14) alkyldimethyl amine oxides, particularly lauryidimethyl amineoxide), non-ionic polyoxyethylene surfactants, e.g., Triton™ X-100 (oroctyl phenol ethoxylate), Lubrol™ PX, Chemal LA-9(polyoxyethylene(9)lauryl alcohol); and glycidols, e.g.,p-sonomylphenoxypoly(glycidol) (Surfactant 10G). A normallynon-denaturing surfactant may function as a denaturing surfactant over aselected concentration range or with respect to certain proteins whichare more sensitive to its denaturing effect than most other proteins.

Non-denaturing surfactant can also include mixtures of non-denaturingsurfactants with denaturing surfactant where the amount of denaturingsurfactant is sufficiently low in the mixture to avoid detrimentaleffect on the protein. Denaturing of a protein by a given surfactant isdependent upon the concentration of surfactant in contact with theprotein and may also depend upon other environmental conditions(temperature, pH, ionic strength, etc.) to which the protein is beingsubjected. The denaturing effects of a selected surfactant, at selectedconcentrations, upon a selected protein can be readily assessed bymethods that are well-known in the art.

Surfactants preferred for use in the preparation of carbon nanotubecomplexes of this invention are dialyzable, i.e., capable of beingselectively removed form a surfactant dispersed carbon nanotubes bydialysis without significant removal of carbon nanotubes or the polymersthat are to be complexed with the carbon nanotubes. Dialyzable,non-denaturing surfactants include, among others, bile acids andderivatives of bile acids, e.g., cholate (salts of cholic acid,particularly sodium cholate), deoxycholate (salts of deoxycholic acid,particularly sodium deoxycholate), sulfobetaine derivatives of cholicacid, particularly3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS);carbohydrate-based surfactants, for example, alkyl glucosides, (e.g.,C6-C14 alkyl glucosides), particularly n-alkyl-β-glucosides (morespecifically, n-octyl-∃3-glucoside (OG)), alkyl thioglucosides, (e.g.,C6-C14 alkyl thioglucosides), particularly n-alkyl-β-thioglucosides(more specifically, n-octyl-β-thioglucoside (OTG)); alkyl maltosides,(e.g., C6-C14 alkyl maltosides), particularly n-alkyl-3-maltosides (morespecifically, n-dodecyl-β-glucoside); and alkyl dimethyl amine oxides(e.g., (C6-C14) alkyldimethyl amine oxides, particularly lauryldimethylamine oxide). Dialyzable, non-denaturing surfactants for use in a givenapplication with a given protein can be readily identified employingwell-known methods.

The term protein is used herein as broadly as it is in the art to referto molecules comprising of one or more polypeptide chains which may belinked to each other by one or more disulfide bonds. Proteins includeglycoproteins (proteins linked to one or more carbohydrates),lipoproteins (proteins linked to one or more lipids), metalloproteins(proteins linked to one or more metal ions) and nucleoproteins (proteinslinked to one or more nucleic acids). The term protein is howeverintended to exclude small peptides, such as those having less than 50amino acids. The term protein includes polypeptides having 50 or moreamino acids. A protein may comprise one or more subunits and thesubunits may be the same or different. For example, a protein may be ahomodimer (having two subunits that are the same) or a heterodimer(having two subunits that are different). Proteins typically have one ormore biological functions. Proteins include enzymes which catalyzereactions and antibodies, transport proteins, receptor proteins or otherproteins which bind to other chemical species (peptides, nucleic acids,carbohydrates, lipids, other proteins, antigens, haptens, etc.).Proteins useful in this invention include soluble proteins, membraneproteins and transmembrane proteins. Soluble proteins are of particularinterest for the formation of carbon nanotube/protein complexes.

The term polypeptide is used to refer to peptides having 20 or moreamino acids and in particular is not intended to refer to peptides suchas those reported in WO 03/102020.

Proteins useful in this invention include those that exhibit selective(or specific) binding to given chemical species or, which are one memberof a set (particularly a pair) of binding partners (e.g., avidin andbiotin, a receptor and a receptor ligand, or an antibody or antibodyfragment and an antigen to which it binds). In specific embodiments,useful proteins include soluble receptors and cell surface receptors. Inother specific embodiments, useful proteins include G-protein coupledreceptors (GPCRs). In more specific embodiments, useful proteins includesteroid receptors, particularly estrogen receptors.

G-protein coupled receptors (GPCRs). are an important and diverse classof pharmaceutical targets in mammalian cellular membranes where theyfunction as signal transducing elements, bind several classes ofbioactive ligands and transmit information to the intracellularmachinery.

In specific embodiments, proteins useful in this invention may containone or more of the carbon nanotube binding sequences disclosed inInternational Patent Application WO03/102020. In another specificembodiment proteins useful in this invention do not contain any one ormore of the carbon nanotube binding sequences disclosed in InternationalPatent Application WO03/102020.

There may be proteins which on complexation with semi-conducting carbonnanotubes form individually dispersed carbon nanotube complexes, butwhich do not exhibit band gap fluorescence. In these cases, the proteinmay itself quench the fluorescence or bind to a species which quenchesthe fluorescence. It has been determined experimentally that serumalbumin (specifically bovine serum albumin, BSA) forms complexes withsemi-conducting carbon nanotubes which do not exhibit band gapfluorescence. It is currently believed that BSA quenches the band gapfluorescence of the carbon nanotube. In a specific embodiment, proteinsof this invention exclude serum albumin, particularly bovine serumalbumin.

There may be proteins, particularly those which tend to self-aggregateor aggregate with other protein, which may on complexation with carbonnanotubes form complexes which exhibit band gap fluorescence, but inwhich the fluorescence is not sensitive to the environment of the carbonnanotube complex. It is currently believed that in such cases, multiplelayers (i.e., more than a monolayer) of protein are formed on the carbonnanotube. The protein can then function to block access to or shield thecarbon nanotube from the environment. Aggregation of proteins complexedwith carbon nanotubes may force the aggregated complexes out of solutionlimiting access of analytes to the carbon nanotube. It has been foundthat concanavalin A, a lectin, forms complexes with semi-conductingcarbon nanotubes which exhibit band gap fluorescence. However, thecarbon nanotube/concanavalin A complexes aggregate and fluorescence ofthe complexed carbon nanotube is not sensitive to the environment of thecarbon nanotube. In a specific embodiment, proteins of this inventionfor forming complexes with carbon nanotubes exclude concanavalin A. Inanother specific embodiment, proteins of this invention for formingcomplexes with carbon nanotubes exclude lectins.

Enzymes function by binding to a substrate and catalyze a reaction ofthe substrate. Substrate selectivity or specificity of an enzyme is, atleast in part, determined by the selectivity or specificity with whichthe enzyme binds to a substrate. Enzymes include among others those thatcatalyze oxidation and/or reduction reactions and those that catalyzecleavage of certain bonds or the formation of certain bonds. It isunderstood in the art that enzyme function may require the presence ofcofactors and/or co-enzymes. Further, it is understood in the art thatenzyme function may be affected by pH, ionic strength, temperature orthe presence of inhibitors. Methods and devices of this inventiontypically employ enzymes which are well-known in the art so that therequirements for any co-factors and/or co-enzymes and the effect of pH,ionic strength, temperature and other environmental factors a well aspotential inhibitors will also be well-known.

Enzymes useful in analyte sensing composition of this invention includeoxidases, dehyrogenases, esterases, oxigenases, lipases, and kinases,among others which may be obtained from various sources. Morespecifically, enzymes useful in analyte sensing compositions includeglucose oxidases, glucose dehydrogenases, galactose oxidases, glutamateoxidases, L-amino acid oxidases, D-amino acid oxidases, cholesteroloxidases, cholesterol esterases, choline oxidases, lipoxigenases,lipoprotein lipases, glycerol kinases, glycerol-3-phosphate oxidases,lactate oxidases, lactate dehydrogenases, pyruvate oxidases, alcoholoxidases, bilirubin oxidases, sarcosine oxidases, uricases, and xanthineoxidases and wherein the analyte is a substrate for the enzyme.

Proteins, including enzymes, useful in this invention can be obtainedfrom various sources, for example, from various commercial sources,through isolation by known methods from natural sources; throughrecombinant methods which are known in the art and by synthetic orsemi-synthetic methods.

Proteins useful in this invention may be truncations, variants,derivatives, or semi-synthetic analogs of a naturally-occurring proteinwhich, for example, has been modified by modification of one or moreamino acids to exhibit altered biological function, e.g., alteredbinding, compared to the naturally-occurring protein, is adeglycosylated form of a naturally-occurring protein or a variant orderivative thereof, or has glycosylation different than that of anaturally-occurring protein. Proteins as well as protein truncations,variants, fusions, derivatives or semi-synthetic analogs ofnaturally-occurring proteins and enzymes useful in this inventionexhibit a biological function that can be used detect an analyte.Protein truncations, variants, fusions, derivatives or semi-syntheticanalogs of naturally-occurring proteins and enzymes may exhibit alteredbinding affinity and/or altered biological function compared tonaturally-occurring forms of the proteins. Protein truncations, forexample, specifically include the soluble portion or portions ofmembrane or transmembrane proteins. Protein fusions, for example,specifically include fusions of the soluble portion or portions ofmembrane or transmembrane proteins with soluble carrier proteins (orpolypeptides).

Enzymes useful in this invention may be a truncation, variant, fusion,derivative, or semi-synthetic analog of a naturally-occurring enzymewhich, for example, has been modified by modification of one or moreamino acids to exhibit altered activity, e.g., enhanced activity,compared to the naturally-occurring enzyme, is a deglycosylated form ofa naturally-occurring enzyme or a variant, fusion, or derivativethereof, has altered glycosylation than that of a naturally-occurringenzyme, is formed by reconstitution of an apo-enzyme with its requiredco-factor (e.g., FAD), is formed by reconstitution of an apo-enzyme witha derivatized co-factor. Enzyme variants, fusions, derivatives orsemi-synthetic analogs of naturally-occurring enzymes may exhibitaltered substrate specificity and/or altered enzyme kinetics compared tonaturally-occurring forms of the enzyme.

Proteins, including enzymes, of this invention can be obtained from anysource organism (e.g., microorganism, bacterium, fungus, animal, orplant). Certain sources may be preferred for use in tissue implantablesensors to avoid adverse or toxic reactions.

The term antibody (or immunoglobulin) as used herein is intended toencompass its broadest use in the art and specifically refers to anyprotein or protein fragments that function as an antibody and isspecifically intended to include antibody fragments including, amongothers, Fab′ fragments. Antibodies are proteins synthesized by an animalin response to a foreign substance (antigen or hapten) which exhibitspecific binding affinity for the foreign substance. The term antibodyincludes both polyclonal and monoclonal antibodies. Polyclonal andmonoclonal antibodies selective for a given antigen are readilyavailable from commercial sources or can be routinely prepared usingmethods and materials that are well-known in the art. A monoclonalantibody preparation can be derived from techniques involving hybridomasand recombinant techniques. Various expression, preparation, andpurification methodologies can be used as known in the art. For example,microbial expression of antibodies can be employed (e.g., see U.S. Pat.No. 5,648,237). Human, humanized, and other chimeric antibodies can beproduced using methods well-known in the art.

An immunoglobulin comprises two heavy and two light chains with theformer being coupled at their hinge region by disulfide linkages. Thetwo heavy chains (but not the light chains) are different for each classof antibody, e.g. IgG, IgM, IgD, IgA and IgE. The distinctions betweenthese classes of antibodies is understood in the art.

Fragments of an antibody can retain the binding affinity of the antibodytoward antigen (or hapten). An immunoglobulin, for example IgG,comprises two heavy and two light chains with the former being coupledat their hinge region by disulfide linkages. Cleavage with papain abovethese linkages releases two antibody binding fragments (Fab) and acrystalline fragment (Fc). Cleavage with pepsin below the hinge resultsin a somewhat smaller Fc fragment and a single F(ab′)₂ fragment with twobinding sites. Each Fab fragment contains both a light chain and part ofa heavy chain, and includes the sequences responsible for specificbinding to an antigen. The Fc portion consists of the remainder of thetwo heavy chains and has effector functions, e.g. relating to bindingand function of complement, macrophages and polymorphonuclear whiteblood cells. As noted above, the two heavy chains (but not the lightchains) are different for each class of antibody, e.g. IgG, IgM, IgD,IgA and IgE.

Fabs are produced from polyclonal or monoclonal antibody preparations.Starting with polyclonal serum or hybridoma supernatant, purifiedimmunoglobulin is digested with papain followed by purification of theFab away from the Fc fragments generated in the digest. Commercial kitsare available such as for preparation of Fab fragments from IgG (PierceProduct No. 44885; Pierce Biotechnology, Rockford, Ill.).

Alternatively, Fab′ molecules are generated by using pepsin digestion ofF(ab′)2 fragments followed by reduction of disulfide linkage between theheavy chains, for example with cysteamine. F(ab′)2 fragments areprepared and isolated by pepsin digestion using art-known techniques andmaterials. Fab′ fragments are then obtained by reduction of the F(ab′)2followed by isolation using art-known techniques and materials. Usingrecombinant techniques, Fab or Fab′ molecules are generated byintroduction of a stop codon in the heavy chain gene at a desiredlocation. For Fab molecules, the location can be within the hinge regionat approximately the codon for the amino acid at which papain digestionoccurs. For Fab′ molecules, the location can approximate the pepsincleavage point. The Fab′ or Fab is then produced directly bysimultaneous expression of both the light chain and engineered heavychain genes to produced their respective proteins which assemble and aresecreted from the cell.

In addition to Fab′ and Fab molecules, other antibody-like molecules,and antibody-derived molecules which retain specific binding ofantibodies can be employed in this invention. For example, single chainantibody variable region fragments (scFv) are employed. Furthermore,hybrid molecules such as bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv) (2) (“tribody”) heterodimers or multimers can be employed(Schoonjans R et al., J. Immunol. 2000 Dec. 15; 165(12):7050-7). scFvcan be prepared with or without disulfide linkages. See Worn A,Pluckthun A., FEBS Lett. 1998 May 15; 427(3):357-61. scFv can beprepared from synthetic or isolated DNA, for example by starting fromthe actual DNA sequence of the desired scFv. An artificial gene usingoligonucleotides is designed, assembled in vitro, and cloned into asuitable expression vector followed by expression in E. coli andpurification of the expressed scFv. Alternatively, scFv are manufacturedfrom monoclonal cell lines. For example, a monoclonal cell line isprovided, and mRNA from the line is cloned to create a cDNA vector fromwhich the variable heavy (V_(H)) and light (V_(L)) chains are thensubcloned into an expression vector.

Other methods for production of antibody fragments are described incurrent editions in the series of Current Protocols titles (allgenerally published by John Wiley and Sons, New York), e.g. CurrentProtocols in Molecular Biology (edited by Frederick M. Ausubel et al.,1991-2004, New York: Greene Pub. Associates and Wiley-Interscience: J.Wiley); Current Protocols in Immunology (edited by John E. Coligan, etal., New York: John Wiley and Sons, 1994-1998).

Sensing compositions of this invention can include carbon nanotubecomplexes with polysaccharides, particularly sensing polysaccharides.The term polysaccharide is used generally herein to include polymers ofany monosaccharide or combination of monosaccharides. A polysaccharidetypically contains 20 or more monosaccharide units. Oligosaccharidecontaining less than 20 monsaccharide units can be used in thisinvention if they are found to complex with carbon nanotubes. Ofparticular interest for assays for monosaccharide analytes are polymersof the monosaccharide analyte (e.g., polymers of glucose for use inassays for glucose). Polysaccharides and oligosaccharides can bederivatized with one or more chemically selective groups or moieties toimpart chemical selectively to the polysaccharide.

Sensing compositions of this invention can include carbon nanotubecomplexes with derivatized polymers that are not proteins,polysaccharides (or oligosaccharides) or other biological polymers suchas polynucleotides. Polymers which complex to carbon nanotubes and areuseful in sensing compositions and methods herein include polymers whichare derivatized to contain one or more chemically selective groups ormoieties which impart chemical selectively to the polymer. Polymers thatcan be usefully derivatized include poly(ethylene glycol), poly(vinylalcohol), poly(vinyl chloride), (e.g., and copolymers thereof, andpolysorbitan esters (e.g., polyoxyethylene sorbitan fatty acid esters.)

The invention relates to sensing compositions, sensing elements whichare adapted to contain sensing compositions, and sensor systems. Asensing element for detecting an analyte comprises a selectively porouscontainer adapted for receiving and retaining the components of asensing composition. The container is sufficiently porous to allowanalyte to enter the container without allowing the functionalcomponents of the analyte sensing composition to exit the container. Thesensing composition is dispersed in a liquid or solid material. Typicalliquids are aqueous solutions which include solutions in which themajority component is water, but which may include alcohols, glycols andrelated water soluble materials that do not affect the ability of thesensing composition to detect or quantitate analyte. The sensingcomposition may be dispersed in a solid matrix. The matrix can be formedfrom various polymers, silica, quartz or other glass, ceramics andmetals with the proviso that the metal matrix is insulated from thesurface with a coating that preserved the optical properties of thecarbon nanotube/sensing polymer complexes. The matrix can be formed froma combination of such solid materials. The matrix can also be asemi-solid material such as a gel or a paste. The matrix must besufficiently porous to allow analyte to enter without loss of sensingcomposition components that are needed to analyte detection. The matrixmust also be sufficiently optically thin or transparent to theexcitation and emission to allow detection of analytes. A solid matrixwith dispersed sensing composition can serve as a sensing element. In apreferred embodiment, the sensing element is an implantable container ormatrix comprising sensing composition which is biocompatible. The term“biocompatible” is employed as broadly as the term is used in the artand in preferred embodiments for human or veterinary applications theterm refers to materials that cause minimal irritation and/or allergicresponse on implantation. The term also preferably refers to materialsin which biofouling of pores is minimized.

Sensing elements include those that are implantable in tissue. Suchsensors may be affected by foreign body encapsulation [31-35] and/ormembrane biofouling of the sensor surface [36, 37]. Fibroblastencapsulation at the site of sensor element implantation has beenreviewed [33. 34] and art-recognized solutions to this problem includeadministration of antigenic factors and anti-inflammatorypharmaceuticals at the site of implantation to promoteneovascularization[31, 36-44]. A sensor surface may be biofouled asendothelial cells adhere and either block or in some cases consumeanalyte [38, 39], thus decreasing the accuracy or otherwise decreasingor destroying the function of the sensor. Sensor architecture can play asignificant role in acerbating or ameliorating the biofouling problem.Biofouling necessarily limits the flux of analyte to the sensor ascellular adhesion becomes more pronounced [45]. Electrochemical sensors,which are the most widely employed for glucose detection, measure theflux of analyte (e.g., glucose) from a limiting membrane. Biofouling insuch sensors immediately decreases the measured signal and is correctedonly by frequent recalibration and eventually replacement is required.In contrast, optical sensors, such as those of this invention, measurethe concentration of analyte at the sensor directly and fouling resultsin a delay in sensor response. A sensor that measures concentrations ofanalyte directly does not exhibit significant distortion of the measuredanalyte concentration until the sensor response rate becomescommensurate with the rate of change in the bulk. Implanted opticalsensors will exhibit an increased stability and longer useful life onimplantation compared to sensors which measure analyte flux such aselectrochemical sensors.

A sensing system for detecting one or more analytes comprises one ormore sensing elements (300) and a detector (310) for measuring anoptical response of the complexes in the sensing solution. An exemplarysensing system is illustrated in FIG. 15. Any appropriate opticaldetector may be employed. The detector can include any and all necessarydevice elements for detecting light and converting the signal detectedinto a form appropriate for analysis or display. Detectors and deviceelements for any needed signal conversion, analysis and display areknown in the art and readily available for use in this invention. It isnoted that the sensing elements of the system may be remote from thedetector. More specifically, the sensing system can include a source ofelectromagnetic radiation (305) to provide electromagnetic radiation(307) of appropriate wavelength for exciting luminescence (315) of thecomplexed carbon nanotube in the sensing composition which can bedetected by the detector. Any known source appropriate for the sensorapplication can be employed including light emitting diodes, or lasers.It is noted that the excitation source may be remote from the sensor andmay also be remote from the detector. In a specific embodiment, thedetector and the excitation source may be combined in a single device.Those of ordinary skill in the art can select light sources and/ordetectors appropriate for use in sensor systems of this invention inview of what is generally known in the art and the specific wavelengthsor wavelength ranges in which the sensor is to operate.

This invention demonstrates a range of new surface assembly and chemicalinteractions whereby selective binding sites can be immobilized on thesurfaces of individually dispersed nanotubes. These sites can then becoupled to the electronic band structure of the carbon nanotube and usedto modulate the optical properties of the nanotube in response tospecific molecular binding events. The term “modulate” is used broadlyherein to indicate any detectible change in an optical property whichcan include a change in intensity of any emission, or any absorption ora change in wavelength of any absorption or emission.

In specific examples, the starting point is an ultrasonicated andpurified (10) solution of HiPco nanotubes suspended according to arecently developed protocol using aqueous surfactant. The chemicalspecies, e.g., a polymer (particularly a sensing polymer) to becomplexed with the carbon nanotube is then combined with the surfactantdispersed carbon nanotubes, and dialyzed against surfactant free buffer.During dialysis as surfactant is removed complexes of the carbonnanotubes with the polymer are formed. The relative amounts of polymerand carbon nanotube are preferably selected to maximized polymercomplexed carbon nanotubes and avoid excess non-complexed polymer. Itmay be desirable in certain cases to select the relative amounts ofpolymer and carbon nanotube to provide complexes which on averagecontain a certain number of polymer molecules complexed to a carbonnanotube. It is preferred that the relative amounts of polymer andcarbon nanotube are selected to achieve monolayer or less coverage ofthe carbon nanotube by the polymer.

In specific embodiments, the polymer is a protein and in this case, anon-denaturing surfactant, such as sodium cholate (2% wt in bufferedsolution), is used in the preparation of the surfactant dispersed carbonnanotubes to prevent denaturing of the protein. The molecule to assembleis then combined with the solution, and dialyzed against surfactant freebuffer (FIG. 1 a.) Within certain concentration ranges van der Waalsforces alone can immobilize a wide range of proteins and enzymes as amonolayer on the nanotube surface, including for example, concanavalinA, avidin, glucose oxidase, and monoclonal mouse anti-human prostatespecific antigen. Above a threshold surface coverage, the processrenders these systems colliodally stable, as evidenced by a preservationof fluorescence(11), and therefore individually isolated (10) even inthe absence of the surfactant phase.

The kinetics of the assembly process can be followed using a combinationof Raman and fluorescence spectroscopies as demonstrated in FIG. 1 b. Inthe case of complexes with glucose oxidase, the system undergoes athermodynamic phase transition after 3.8 hours upon slow removal of thesurfactant as glucose oxidase is assembled along the nanotube surface.The emission maximum of the (9,1) nanotube is shown to decrease inenergy by 10 meV, indicating that the tightly packed cholate adsorbedphase has been replaced by a more porous enzyme layer. The increasedaccessibility of water and the resulting polarity at the surface of thenanotube decreases the energy of the excited state and causes thissolvatochromic shift (13).

These gaps between absorbed enzymes and proteins provide sections ofexposed nanotube surface whose reactivity and electron transfercharacteristics can be explored in the absence of an interfering(typically charged) surfactant layer (10, 11, 13, 16, 17) foressentially the first time. We show that non-covalent electron transferat the nanotube surface can introduce functional groups with apreservation of electronic structure. In FIG. 2 a, the exposed surfaceis titrated using ferricyanide ions (Fe(CN)₆ ⁻³) which form a chargetransfer complex at the nanotube thereby localizing electrons andshifting the Fermi level into the valence bands. The process can bemonitored optically as the transition decays with increasingfunctionalization. Saturation of the exposed surface occurs atapproximately 11.3 ions/unit cell length and attenuates the fluorescencefrom the sample by 83.3% (FIG. 2 b). The surface modification isirreversible: we find that it is stable to dialysis against pure bufferindefinitely with no restoration of fluorescence or desorption back intosolution. However, the absence of an increase in the nanotube disordermode in the Raman spectrum indicates that no covalent bonds on thecarbon surface have been broken(1, 2). A number of electroactive speciescan be immobilized in this way and used to modify the surface fordesired functionality. FIG. 2 b also shows the behavior of the reductionproduct (19) ferrocyanide (Fe(CN)₆ ⁻⁴) at comparable loading. We notethat the reduced electron affinity of this complex results in a lessperturbing electron withdraw and only a 27.4% attenuation offluorescence.

These insights offer new routes for the chemical modification ofindividual nanotubes in solution in a manner that exploits, but does notdisrupt, their electronic properties. For example, the electroactiveferricyanide layer can act as an enzyme mediator(19), shuttlingelectrons to and from β-d-glucose reaction at the enzyme layer. FIG. 3 ais a molecular simulation of such a bifunctional nanotube substrateshowing the coupling between the reaction at the enzyme and the electrontransfer to the nanotube surface. As β-d-glucose is oxidized to thed-glucono-1,5-lactone, the H₂O₂ co-product reduces the mediator, whichelevates the Fermi-level of the nanotube with a restoration offluorescence. Hence, the substrate couples the binding event at theenzyme to electron transfer at the nanotube surface and modulates thefluorescence in a quantitative manner.

The practical utility of such a structuring of the nanotube surface isdemonstrated by loading the resulting solution into a sealed 200 μmdiameter dialysis capillary (13 kDa molecular weight cut-off.) Here, thetarget analyte is free to diffuse across the capillary boundary whilethe sensing medium (nanotubes with an average length of 1.5 μm) isretained. When excited with a 785 nm photodiode laser, the fluorescentemission of the (6,5) nanotube (λ_(max)=1041 nm) is shown to respond tothe local glucose concentration after an 80 s transient, even in astrongly absorbing blood specimen(21). We find that adjusting the enzymeto redox mediator ratio at the nanotube surface allows for a tuning ofthe response into the range needed to monitor blood glucose in diabeticpatients (1 to 8 mM), for instance. This tuning is done at the expenseof overall sensitivity, but devices responsive in the desired range havebeen made with as low as 34.7 μM for the detection limit of glucose, andthis corresponds to 2.2 molecules detected per nm of nanotube length.The response function (FIG. 4) relating the normalized intensity toglucose concentration follows a Langmuir adsorption profile with bindingconstant of 0.92 (mM⁻¹). The advantage of the near infrared signaling toand from such a capillary device is its potential for implantation intothick tissue or whole blood media, where the signal may penetrate up toseveral centimeters. Such a passive, optically responsive substrate mayallow the realization of continuous analyte detection(9) in-vivo usingan external, miniaturized excitation and detection device. Few organicmolecules fluoresce in the wavelength range necessary for in-vivodetection, and those that do so invariably lack long termphoto-stability(20). The methods described herein allow multiplefunctionalities to be introduced onto singly dispersed nanotubes insolution such that they retain the near infrared fluorescence. Themethods herein are useful in synthesizing nanotube-based optical sensorsand are further useful for preparing image contrasting agents, activebio-markers or electrodes where an isolated nanotube is desired, and theintrinsic, 1-D electronic structure must be preserved.

When a group of substituents is disclosed herein, it is understood thatall individual members of those groups and all subgroups, including anyisomers and enantiomers of the group members, and classes of compoundsthat can be formed using the substituents are disclosed separately. Whena compound is claimed, it should be understood that compounds known inthe art prior to the invention herein, including the compounds disclosedin the references disclosed herein, are not intended to be included inthe claim. When a Markush group or other grouping is used herein, allindividual members of the group and all combinations and subcombinationspossible of the group are intended to be individually included in thedisclosure.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.Specific names of compounds used herein are intended to be exemplary, asit is known that one of ordinary skill in the art can name the samecompounds differently. When a compound is described herein such that aparticular isomer or enantiomer of the compound is not specified, forexample, in a formula or in a chemical name, that description isintended to include each isomers and enantiomer of the compounddescribed individual or in any combination. Whenever a range is given inthe specification, for example, a temperature range, a time range, or acomposition range, all intermediate ranges and subranges, as well as allindividual values included in the ranges given are intended to beincluded in the disclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, can beexchanged with “consisting essentially of” or “consisting of”.

One of ordinary skill in the art will appreciate that methods, apparatusand materials, including among others, device elements, startingmaterials, reagents, synthetic methods, purification methods, analyticalmethods, and spectroscopic methods, other than those specificallyexemplified can be employed in the practice of the invention withoutresort to undue experimentation. All art-known functional equivalents,of any such methods, apparatus and materials are intended to be includedin this invention.

Although the description herein contains many specificities, theseshould not be construed as limiting the scope of the invention, but asmerely providing illustrations of some of the embodiments of theinvention. All references cited herein are hereby incorporated byreference in their entirety. Some references provided herein areincorporated by reference herein to provide details concerningadditional starting materials and additional methods of synthesis ofcarbon nanotubes, additional methods of purification of carbonnanotubes, sources of proteins, for use in the invention, additionalsurfactants for use in the invention, additional polymers for use in theinvention, more details of method for characterizing carbon nanotubesand carbon nanotube complexes, additional methods of analysis andadditional uses of the invention.

The following examples are intended to further illustrate and describethe invention but are not intended to limit the invention.

THE EXAMPLES

Glucose oxidase immobilization on SWNTs. Single walled carbon nanotubes(SWNT) product (7 mg/L) from a high-pressure CO reactor (HiPco) (RiceUniversity research reactor run 107), were suspended in H₂O with 2% wt.sodium cholate (Sigma Aldrich). The suspension was prepared usinghigh-shear mixing, sonication and centrifugation as previously described(10) with the expectation that that 1% SDS was replaced with 2% sodiumcholate. Sodium cholate is a surfactant that does not denature theprotein.

Glucose Oxidase (GOx) from Aspergillus niger (Sigma Aldrich) was addedto the cholate suspended SWNT (10 mL), to obtain a ratio of 66 mg GOx/mgSWNT in the mixture. Three milliliters of the GOx/cholate/SWNT mixturewas added to a dialysis cartridge (Pierce), which was then placed into 2liters of standard Tris buffer at pH 7.4 and dialyzed overnight. Theamount of added glucose oxidase was chosen to yield a monolayer on thenanotube surface and minimize the amount of possible free GOx in themixture that is not associated with the nanotube surface. (The preferredratio of GOx/SWNT was empirically determined based on the phase diagramdiscussed below or alternatively can be estimated based on thedimensions of the protein.)

The composition of the starting solution was chosen to minimize freeenzyme not adsorbed on the nanotubes. The molar ratio is (0.066 gGOx)/(160 kDa dimer GOx) (12 Da carbon)/(0.001 g nanotubes) or 1 dimerof GOx per 200 carbon atoms. For a (10,10) nanotube, this isapproximately 1 dimer on either side of the nanotube every 10 nm ofnanotube length.

FIG. 5 is a graph showing transient fluorescence measurements of theSWNT/cholate/GOx mixture (upper line) and a control dialysis sample ofthe SWNT/cholate suspension (lower line) as a function of dialysis time.Fluorescence at 785 nm excitation was normalized to the Raman tangentialmode at 1592 cm⁻¹. Fluorescence was measured using a CCD camera. Asdiscussed above for FIG. 1B, normalized fluorescence decreases ondialysis of the SWNT/cholate/GOx mixture with a clear transitionoccurring at about 3.8 hrs. Ultimately fluorescence intensity isdecreased by a factor of about 2.2. The control experiment was performedusing identical procedures without the addition of the GOx. For thecontrol suspension carbon nanotube fluorescence decays relativelyrapidly to zero and small particles containing flocculated nanotubes arevisible in the sample.

The relative stability of the SWNT/GOx suspension and the SWNT/cholatesuspension after dialysis can be ascertained by visual examination. TheSWNT/cholate suspension and the dialyzed SWNT/GOx suspension aretranslucent amber with no visible flocculation. In contrast, uponremoval of the cholate by dialysis, the once dispersed nanotubes formlarge aggregates. If the protein is present in the dialyzed SWNT/cholatemixture above a critical concentration, the mixture is a well-dispersedsuspension which is stable.

Adsorbed phase diagram. FIG. 6 is a graph of normalized fluorescence asa function of the weight ratio of GOx (protein) to SWNT inSNWT/cholate/GOx samples after dialysis (approximately 20 h). Loweringthe starting concentration below the threshold used for the aboveexperiments results in a flocculated system. Using nanotube fluorescenceand its absence upon aggregation, a phase diagram can be made for thesystem being studied. Here, the measured fluorescence is normalized bythe intensity of the tangential Raman mode measured at 785 nm.

Based on these data it was determined that a GOx to SWNT ratio of about66:1 or higher is preferred to solubilize all of the nanotubes in thesample and to minimize the amount of free enzyme in solution.

The UV/near infrared absorption spectrum of the GOx/SWNT complex (at 37C) is shown in FIG. 7 (spectrum a). After addition of 48 mM ferricyanide(spectrum c) E11 transitions above 1100 nm decrease, while transitionsbetween 900 and 1100 nm remain unchanged. The apparent increase inabsorption between 500 and 700 nm is due to ferricyanide absorption inthe visible. The solution was then titrated with 2 mM hydrogen peroxide(spectrum b) resulting in only partial restoration of the longestwavelength excitations and no change in the other spectral features.

SWNT/GOx Sensor Testing. A reactor setup was used to test the SWNT/GOxglucose sensor. The sensing medium, GOx-SWNT or the control 2% wt.cholate-SWNT, was loaded into a dialysis cartridge (having a 10 KDamolecular weight cutoff) and placed in TRIS (pH 7.4) buffer in areaction chamber. A temperature controller with a heating coil and athermocouple was used to maintain temperature at 37° C. A peristalticpump was used to cycle reagent-free buffer into and remove buffer fromthe reaction chamber. The fluorescent emission of nanotubes was detectedusing a long-distance objective lens via a thermoelectrically-cooled CCD(Andor) with 785 nm excitation from a photodiode laser.

Aliquots of 0.5 M potassium ferricyanide solution were added directly tothe stirred buffer in the reaction chamber to adjust the concentrationof ferricyanide therein. Potassium ferricyanide, is free to diffuseacross the membrane, while the sensing medium (SWNT/GOx) containingnanotubes is retained in the cartridge. FIG. 8 is a graph showing theeffect of additions of ferricyanide to the sensing medium by dialysisinto the dialysis cartridge. The fluorescence of GOx-SWNT diminishes inresponse to ferricyanide addition. Cycling in reagent free buffer(dropping ferricyanide concentration in reaction chamber to zero)results in only a partial restoration of fluorescence intensity. It isbelieved, as discussed above, that a portion of the ferricyanide ischemisorbed to SWNT/GOx complexes and is not removed. The sameexperiment was performed with a control of 2% wt. cholate suspendedSWNTs. No diminution in fluorescence was observed on addition offerricyanide.

Glucose Sensing. A SWNT/GOx suspension prepared as described abovehaving a 66:1 weight ratio of GOx:SWNT (600 microL) was introduced intoa cuvette maintained at 37 C. The sample was excited using a 785 nmlaser (power=35 mW) and the resultant near-infrared (nIR) fluorescentlight was scattered back 180° and recorded using a nIR sensitive CCDcamera. While continuously monitoring the nanotube fluorescence, 100microL of the potassium ferricyanide solution (0.5 M) was added to thecuvette (62.5 mM) leading to a fluorescence decrease. Once thefluorescence reached a steady state value, 4 microL of glucose (1.2 mM)in 0.5 M phosphate buffer (pH 7.4) was added, giving a total initialglucose concentration of 1.42 mM in the cuvette. This process wasrepeated twice more, each time adding a 4 microL injection of glucose toincrease the glucose concentration in the cuvette to 4.2 mM and 7.0 mM,respectively. As shown in FIG. 4C normalized fluorescence intensityincreased with addition of increasing concentration of glucose.

Glucose Sensing Control Experiments. Control glucose sensing experimentswere performed using 2% cholate suspended SWNT and GOx-SWNT withoutaddition of ferricyanide. These suspensions were both buffered (pH 7.4)and maintained at 37 C. As shown in FIGS. 9A and 9B, no significantchange in fluorescence was observed on glucose addition in these controlexperiments.

Glucose Sensing in Pig Serum. GOx suspended SWNT (GOx:SWNT weightratio=66:1) was injected into a dialysis capillary (Spectrum) with a 13kDa MW cutoff and the dialysis capillary was inserted into a smalldiameter, ˜12 mm, glass capillary. The dialysis capillary was closedusing a cyanoacrylate adhesive. One end of the glass capillarycontaining the dialysis capillary was inserted into 0.5 M phosphatebuffer (pH 7.4) at 37 C, causing the buffer to fill the glass capillary.Laser light of 785 nm (35 mW) was focused onto the capillary with thefluorescence emission being scattered back 1800 through a notch filterilluminating a CCD camera used to record the signal. Approximately 1 mLof a 62.5 mM potassium ferricyanide solution in phosphate buffer (pH7.4, warmed to 37 C) was then injected into the glass capillarydisplacing the buffer therein and causing ferricyanide to diffuse intothe dialysis capillary with a resultant decrease in nanotubefluorescence. The solution in the glass capillary surrounding thedialysis capillary can be replaced with ferricyanide-free buffer with norestoration of the fluorescence as discussed above. Pig serum from apregnant female warmed to 37 C, was then injected into the glasscapillary allowing serum glucose to diffuse through the membrane andregister a response.

Determination of GOx Kinetic Constants. To determine GOx kineticconstants under the sensing conditions, 600 microL of GOx (0.0127 mM) in0.5 M phosphate buffer (pH 7.4) and 100 microL of the 0.5 M ferricyanidesolution (in 0.5 M phosphate buffer, pH 7.4) were well mixed andmaintained at 37 C in a jacketed cuvette. The sample was illuminatedwith a 785 nm laser and the resultant ferricyanide Raman scattering fromthe mediator, evident at 2132 cm⁻¹, was collected at 1800 and recordedby a CCD camera. While continuously monitoring the ferricyanide Ramanscattering peak, 100 microL of glucose solution (20 mM) was added to thecuvette. The oxidation of glucose by glucose oxidase caused hydrogenperoxide to be produced, which then reduced the free ferricyanide,Fe(CN)₆ ³⁻, to ferrocyanide, Fe(CN)₆ ⁴⁻, resulting in an intensity lossfrom ferricyanide scattering. Ferricyanide scattering intensity wasnormalized to one by dividing the specific intensity by the initialintensity. FIG. 10 shows the change in normalized ferricyanidescattering intensity monitored via Raman scattering at 785 nm at 2132cm⁻¹ over time after addition of 20 mM glucose. The same experiment wasalso completed for glucose concentrations of 31, 10 and 5 mM. Theferricyanide response was fit with a normalized ferricyanideconcentration, C/C_(o), modeled using Michaelis—Menton kinetics$\frac{I}{I_{0}} = {{\frac{C}{C_{0}} \approx \frac{\mathbb{d}\lbrack G\rbrack}{\mathbb{d}t}} = \frac{{k_{2}\left\lbrack {G\quad O\quad x} \right\rbrack}\lbrack G\rbrack}{K_{m} + \lbrack G\rbrack}}$

where I₀ and I are the initial and transient ferricyanide scatteringintensity, [G] is the glucose concentration, [GOx] is the total enzymeconcentration, K_(m) is the Michaelis-Menton constant and k₂ is thereaction rate constant. Fitting all four data sets yields kineticconstants K_(m)=4.21 mM and k₂=293.4 min⁻¹. The fit of the model for oneresponse can also be seen in FIG. 10.

Determination of GOx Activity in the GOx/SWNT Complex. Nanotubefluorescent data from sensing glucose in buffer was used to determinethe effective GOx concentration under identical conditions as above.Fluorescence intensity was normalized to (I−I_(b))/(I_(a)−I_(b)) where Iwas fluorescence intensity, I_(a) was fluorescence intensity of pristinenanotubes and I_(b) was fluorescence intensity after the ferricyanideadsorbed to the nanotube surface. The normalized intensity was then usedin conjunction with the glucose response function to calculate glucoseconcentration for each data point. Using the previously calculated K_(m)and k₂, the effectiveness factor, defined as the effective GOxconcentration divided by the actual GOx concentration, was calculated.The change in glucose concentration was again described usingMichaelis-Menton kinetics, and the GOx concentration was modified togive the best fit to the data. This modified GOx concentration was thenconsidered the effective GOx at the SWNT sidewall. The method of leastsquares was used to minimize error between the calculated andexperimental glucose concentrations. The fit of the first glucoseinjection during sensing in buffer can be seen in FIG. 11 (the linerepresents the model and the squares are the data).

A comparison of these responses reveals no loss of GOx enzyme activitywhile attached to the carbon nanotube surface. Hence, the enzyme is notdenatured or inactivated, but quite stable despite the dialysis process.

AFM Analysis of GOx-SWNT and controls. Tapping mode atomic forcemicroscopy (AFM) of glucose oxidase and glucose oxidase-suspendednanotubes (GOx-SWNT) was performed with a Digital Instruments MultimodeIIIa Scanning Probe Microscope (Veeco Instruments, Woodbury, New York)using 100 um v-shaped cantilevers with oxide-sharpened silicon nitridetips (Veeco Metrology LLC., Santa Barbara, Calif.) Samples weresuspended 10 mM Tris buffer at a pH of 8 with 0.15 M NaCl and 10 mMMgCl₂ and deposited onto a mica surface. Wet AFM images were made inbuffer. Review of these images indicates an average height of 4.4 nmsuggesting monolayer coverage of glucose oxidase on the carbonnanotubes.

Control AFM images of HiPco nanotubes were taken in tapping mode with aDigital Instruments 3100 Scanning Probe Microscope using BS-Tap300Alaluminum-coated silicon tips (Budget Sensors, Sofia, Bulgaria).Nanotubes suspended in 100 mM sodium cholate were deposited onto asilicon wafer coated with 3-aminopropyltriethoxy-silane and rinsed after20 seconds. Images were taken on the dried surface. Height measurementsof these carbon nanotubes are typically between 0.6 and 1.5 nm.

Detectibility of SWNT near-IR fluorescence through a Tissue Sample. Acapillary loaded with GOx suspended SWNT was placed underneath a sampleof cultured human epidermal keratinocytes (MatTek). An area map measuredat 20× magnification monitoring nanotube fluorescence at 785 nmexcitation clearly shows an image of the capillary through the tissuesample. Fluorescence of a semiconducting carbon nanotube is readilydetectible though the tissue sample.

Estradiol-17β(E2) Sensor. FIG. 12 illustrates an estradiol-17β(E2)sensor in which a sensing polymer covalently derivatized with one ormore E2 molecules (antigens) or E2 derivatives (antigen derivatives) isnon-covalently complexed with carbon nanotubes, specifically withsemiconducting carbon nanotubes which exhibit band gap fluorescence. Asensing solution comprises carbon nanotube/sensing polymer complexes andantibody that specifically binds to the antigen and the covalentlylinked antigen or antigen derivative. The analyte detected is theantigen. As free (unbound) antigen comes into contact with the analytesensing composition, it binds to free antibody (in equilibrium withbound antibody) and shifts the equilibrium of antibody bonded to thecovalently attached and free antibody toward free antibody which isdetected by as an increase in near IR fluorescence of the carbonnanotube. The sensing polymer may be a polymer derivatized with one ormore antigens or antigen derivatives (the antibody must have someaffinity for binding to the antigen derivative.) The concentration ofthe antigen in contact with the sensing solution is determined byfollowing changes in fluorescence (intensity or wavelengths shift.) Forexample, poly(ethylene glycol) can be functionalized with a derivativeof 17β-estradiol, e.g., 3-(O-butyric acid)estradiol using a proceduresimilar to that described in the BIAcore system Manual, BIAcore UppsalaSweden 1991 for treatment of Surface Plasmon Resonance surfaces.Alternative derivatization methods are known in the art.

As an alternative, the sensing polymer can be a protein scaffold withestradiol or estradiol derivatives covalently attached thereto.

As illustrated in FIG. 12, an analyte sensing solution containing thecarbon nanotube complex with the sensing protein and containing antibodyis introduced into a dialysis capillary to form a sensor element. Asshown, the sensor element may be implanted in tissue or contacted with abiological fluid to assess levels of E2 in the tissue or biologicalfluid. The sensing composition components illustrated in FIG. 12 can beemployed to detect antigens including steroids, other than estradiol.

The illustrated E2 sensor can be employed, for example, for trackingestrus in an animal, particularly in cows and pigs. Measurement of E2levels will allows improved timing of insemination of animals and can beused to monitor pregnancy in the animals. Sensors for the detection ofthe presence of and concentration of other steroids in tissue orbiological fluids can be made in an analogous manner.

Preparation of Avidin/Carbon Nanotube Complexes. Avidin Suspended SWNTwere prepared using dialysis as described above. SWNT decant suspendedin 2% cholate and 1×Tris buffer were combined with avidin in a 3 mLdialysis cassette. The cassette system was dialyzed once for 17 hours in500 mL of lightly stirred 1×Tris buffer, then dialyzed again for 14hours in 2 L of the buffer. Spectra of the initial and final solutionswere taken using fluorescence and Raman scattering to confirm that theSWNT did not bundle during the dialysis process. Avidin suspended SWNTcontaining individually dispersed carbon nanotubes which exhibited bandgap fluorescence. A commercial avidin preparation was employed.Streptavidin or avidin from egg white can be employed. Additionallyvarious labeled avidins can be employed.

Biotin interaction with the avidin suspended SWNTs was probed usingfluorescence and Raman scattering. The avidin SWNT suspension waspipetted into a quartz cuvette, and stirred while continuous spectrawere taken every 3.3 min, monitoring the area under the fluorescencepeak for the (6,5) nanotube (see FIG. 14.) After 30 minutes, 2 μL ofbiotin in water (0.22 μg/μL) were added to the avidin SWNT system,inducing a gradual decline in the fluorescence peak area. Fouradditional aliquots of 2 μL biotin were added in 1 hour intervals. Thesecond addition of biotin produced a 50% drop in the peak area within6.5 minutes. However, the following biotin addition caused a 25%recovery of the fluorescence within 3.3 minutes, then a slow steadydecrease. Subsequent biotin additions resulted in an immediate increasein fluorescence, followed by a slow decline of the peak area. Theseresults demonstrate that fluorescence emission of avidin suspended SWNTsare sensitive to binding of biotin and can be employed for sensingapplications on the nanomolar level. Avidin suspended SWNTs whichcontain avidin complexed with single-walled carbon nanotubes exhibitnear IR fluorescence and are particularly useful in implantable sensors.Avidin suspended SWNTs can be used to detect biotin and various biotinderivatives as well as biotinylated species. Avidin suspended SWNTs canbe used to detect various chemical and biological species that have beensubjected to biotinylation. Biotinylation kits are available fromcommercial sources for a variety of applications.

An Optical Glucose Sensor Based on Competitive Binding. A reversibleglucose sensor using SWNTs as the fluorophore that is based oncompetitive binding is illustrated in FIGS. 14A and B. FIG. 14 Aillustrates a sensing element particularly for implantation into tissue.FIG. 14 B illustrates the mechanism of the competitive binding that isthe basis of the sensor. Nanotubes are complexed or coated in a polymerthat functions as a glucose analog, such as dextran, for binding to orreaction with a glucose-specific protein. Specifically, the sensingcomposition comprises SWNT/dextran complexes, and a known concentrationof the a protein that binds selectively to glucose (and the dextranpolymer). Specific examples of such proteins include apo-glucoseoxidase, which retains glucose binding function, but not its enzymeactivity, or the glucose-binding lectin, concanavalin A (ConA). Thesensor element of FIG. 14A is shown for implantation and employs abiocompatible hydrogel which optionally comprises growth factors such asVEGF for release into the surrounding tissue.

Binding of protein to the polymer (dextran) at the surface of thenanotube attenuates SWNT fluorescence, which is reversed by theintroduction of glucose into the system (FIG. 14B). The sensingcomposition containing the SWNT/dextran complex and the glucoseselective protein is then introduced into a dialysis capillary with amolecular weight cutoff such that the sensing composition is retainedand glucose can freely diffuse across the barrier.

The capillary is preferably coated with a porous, biocompatiblehydrogel, such as described in [48-51] to reduce or preventencapsulation and detriment to the sensing device. Growth factors, suchas vascular endothelial growth factor (VEGF), can be imbedded in thehydrogel matrix and released in a controlled manner to inducevascularization [48] around the capillary. Once implanted beneath theskin excitation of the sample can be accomplished using a laser diodecoupled with an InGaAs detector array, as illustrated in FIG. 15. Thesensor system illustrated in FIG. 15 can be employed for a variety ofsensing compositions of this invention.

To understand the sensor response, the equilibrium response of thesensor can be modeled. The total amount of binding protein bound to thesurface of the nanotube can be determined using the equilibrium bindingconstants for the two competitive reactions,K _(G)=[Protein-Glucose]/[Protein][Glucose]K_(SWNT)[Protein−SWNT]/[Protein][SWNT]

where KG is the equilibrium binding constant for binding protein andglucose, KSWNT is the equilibrium binding constant for binding proteinand dextran coated SWNT, [Protein−Glucose] and [Protein−SWNT] are theconcentrations of binding protein bound to glucose and SWNT,respectively, and [Protein] and [Glucose] are the concentrations of eachunbound species. Finally, [SWNT] is the total concentration of unboundprotein binding sites on the surface of the nanotube. To estimate thetotal number of binding sites on the surface of the nanotube, reasonableassumptions are that the nanotube concentration is 50 mg/L, eachnanotube is ˜1 μm long, and that there is a protein binding site every10 nm along the SWNT surface, giving a binding site concentration of ˜10μM. Because proteins localized near the surface of the nanotubeattenuate SWNT fluorescence, the normalized fluorescence from the sensorcan be modeled as (Total Surface-Occupied Surface)/Total surface.Finally, using equilibrium constants for ConA as an estimated (KG=400L/mol) and an KSWNT of 15000 L/mol [50], SWNT fluorescence can bemodeled for different protein concentrations. Modeling indicates thatsuch a glucose sensing system is useful for detection of biologicallyrelevant glucose concentrations (2-30 mM) and that the sensor responsecan be tuned based solely on protein concentration. Modeling indicatesthat practically useful levels of performance and sensor response can beobtained for reasonable protein concentrations in the range of tenths ofmM.

Use of a Functionalized Polymer as a Sensing Polymer. Tween 20™ (apolyoxyethylene sorbitan fatty acid ester, specifically olyoxyethylenesorbitan monolaurate ester) was functionalized with biotin (biotin-longchain-PEO-amine) employing 1,1 carbonyldiimidazole (CDI) (51). Thebiotinylated Tween 20 (BioTween) was then immobilized on the surface ofthe nanotube using the dialysis procedure described above. The resultingnanotube solutions were free of visible aggregates and were stablestored at 4 C for >3 months.

Functionalization of Tween 20 for Biospecificity: Tween 20 was reactedwith CDI in DMSO (dried with molecular sieves) for 2 hr at 40° C. withmixing under a N₂ blanket. The product was precipitated using diethylether, and the collected material was redissolved and precipitated twicemore to ensure removal of reaction byproducts. To add a biotinfunctional group to Tween, the CDI functionalized Tween was allowed toreact with biotin-long chain-PEO-amine in standard Tris buffer (pH 7.4)for 24 hr at room temperature with mixing. Tween 20 andcarbonyldiimidazole (CDI) were purchased from Sigma Aldrich (St. Louis,Mo.). Biotin-long chain-polyethylene oxide (PEO)-amine was purchasedfrom Pierce.

Suspension of SWNT with Functionalized Tween. Single walled carbonnanotubes were suspended in 2 wt % sodium cholate in nanopure H₂O usinga high intensity sonication method previously demonstrated to yieldindividually dispersed nanotubes. Functionalized Tween was added to thenanotube suspension in the ratio of 1:3. The resulting mixture wasplaced into a 10,000 molecular weight cutoff dialysis cassette anddialyzed against surfactant free buffer for 24 hrs, with the bufferbeing replaced at the 4 hr mark. The resulting solution shows no signsof aggregation and is stable for >1 month. The single walled carbonnanotubes used in this study were obtained from CNI, reactor run 107. Tomeasure nanotube fluorescence, individually dispersed single walledcarbon nanotubes were excited with a 785 nm photodiode laser (Invictus)and the resulting fluorescence emission was collected at 180° through anotch filter and shown onto a thermo-electrically cooled nIR CCD camera(Andor)

Changes in the SWNT photoluminescence during the removal of cholate bydialysis and the subsequent assembly of BioTween on the surface of thenanotube was monitored using a 785 nm photodiode laser. The fluorescencespectra obtained before and after a 20 hr dialysis against surfactantfree buffer show that nanotube fluorescence decreases and emission fromthe (6,5) nanotube exhibits a shift to lower energy. Monitoringfluorescence decrease as a function of time shows a gradual decreasewith time. However, closer inspection of the fluorescence data showsthat both features do not decrease at the same rate. Emission from the(6,5) nanotube decreases at a faster rate than that of the (7,5)nanotube. This effect saturates at approximately 10 hrs.

As a control, Tween 20 functionalized with 1,1 carbonyldiimidazole wasallowed to react with water, creating an ionic Tween surfactant(HydroTween). This HydroTween was also assembled on the surface of thenanotube while monitoring SWNT optical properties. Assembly of theHydroTween on the surface of the nanotube shows similar behavior to thatof the BioTween, with nanotube fluorescence decreasing during assemblyand the surfactant showing similar selectivity between (6,5) and (7,5)nanotube species. However, in the case of assembly of HydroTween theselective decrease effect saturates after only 4 hrs. These resultsindicate that the biotin moiety does not act as a fluorescence quencher.However, it appears that biotin plays a role in the assembly process asevidenced by the differences in assembly time observed for complexes ofBioTween and HydroTween. Attempts to suspend nanotubes withnon-functionalized Tween 20 were unsuccessful, likely due to thenon-ionic nature of the non-functionalized surfactant. It is believedthat the end groups of Tween 20 are not sufficiently long to preventSWNT-SWNT interactions from occurring. It was also found that highlyfunctionalized samples of BioTween were also unsuccessfully forsolubilization and suspension of SWNT. Tween 20 functionalized with CDIhas three potential sites for further functionalilzation. Partialfunctionalization of Tween 20 with biotin leaves some remainingcarboxylic acid groups. The presence of these carboxylic acid groups isbelieved to impart a partial negative charge to the surfactant causingcharge-charge repulsion and sufficient hindrance to prevent nanotubetube aggregation.

Streptavidin binding to the BioTween —SWNT complex. Total internalreflection (TIR) fluorescence microscopy, capable of single moleculefluorescence measurements, was employed to assess streptavidin bindingto the BioTween-SWNT complex.

To first determine if the biotin in the BioTween/SWNT complex was stillavailable for binding, a streptavidin coated surface was prepared bycontacting biotinylated BSA immobilized non-specifically on a surfacewith streptavidin. The streptavidin coated surface was then washed witha dilute BioTween/SWNT solution. If the biotin in the complex remainscapable of binding to streptavidin, nanotubes should remain immobilizedon the surface. The treated surface potentially having immobilizedBioTween/SWNT complexes was washed with quantum dots coated instreptavidin. Fluorescence microscope measurements show the presence oflinear nanotube-like structures indicating that nanotubes are bound tothe surface. A control surface treated with BioTween without nanotubeswhich was washed with quantum dots coated in streptavidin showed nofluorescing linear structures. These results indicate that the biotin inthe BioTween/SWNT complex remains free to bind to biotin bindingpartners. (e.g., streptavidin and avidin).

Upon the addition of 2.02 microM streptavidin to the BioTween-SWNTcomplex, a significant decrease in fluorescence from the (6,5) nanotubewas observed. This decrease occurred over 1-2 h, with an increase in thenoise occurring after ˜0.5 hr. Streptavidin is a tetramer with 4 biotinbinding sites, the increase in the noise is believed to be due tonanotube aggregation induced by multiple binding events. The noise isdecreased by shear mixing of the solution, consistent with disruption ofaggregation. However, after a sufficient amount of time, theBioTween-SWNT-streptavidin aggregates reform. If streptavidin bindingsites are partially saturated with biotin prior to addition toBioTween-SWNT a fluorescence decrease is still observed. The resultsindicate that unblocked streptavidin can bind to more than oneBioTween/SWNT complex.

The change in fluorescence of the BioTween/SWNT complex changes as afunction of the amount of protein (streptavidin or avidin) added to asensing composition containing the complex. Increasing proteinconcentration results in larger decreases in fluorescence. In contrast,addition of bovine serum albumin (BSA), a protein which is known toexhibit a high degree of non-specific binding to surfaces, to theBioTween/SWNT complex caused no fluorescent response. Thus, the observedchanges in fluorescence are due to specific binding of the protein tothe biotin moiety of the complex rather than to non-specific binding ofprotein to the nanotube.

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1. An analyte sensing composition for detecting an analyte, ordetermining the concentration of the analyte, which comprises a sensingcomplex of a sensing polymer with a semi-conducting, single-walledcarbon nanotube, wherein the carbon nanotube of the sensing complexexhibits photo-induced band gap fluorescence in the near-infrared, thesensing polymer selectively binds to or selectively reacts with theanalyte, and the photo-induced band gap fluorescence of the carbonnanotube of the sensing complex is responsive to the selective bindingof the sensing polymer to the analyte or the selective reaction of thesensing polymer with the analyte.
 2. The analyte sensing composition ofclaim 1, wherein the sensing polymer is a protein.
 3. The analytesensing composition of claim 1, wherein the sensing polymer is an enzymewhich selectively reacts with the analyte.
 4. The analyte sensingcomposition of claim 1, wherein the analyte is glucose and the sensingpolymer is a glucose oxidase.
 5. The analyte sensing composition ofclaim 1, wherein the sensing polymer is an antibody or antibody fragmentand the analyte is an antigen which binds to the antibody or antibodyfragment.
 6. The analyte sensing composition of claim 5, wherein theantibody is a monoclonal antibody.
 7. The analyte sensing composition ofclaim 1, wherein the analyte is an antibody or antibody fragment and thesensing polymer is a protein comprising one or more epitopes which bindto the antibody of antibody fragment.
 8. The analyte sensing compositionof claim 1, wherein the sensing polymer is a polysaccharide.
 9. Theanalyte sensing composition of claim 1, wherein the sensing polymer iscovalently bonded to the analyte, or a derivative or analog thereof. 10.The analyte sensing composition of claim 9, wherein the sensing polymeris a protein.
 11. The analyte sensing composition of claim 9, whereinthe sensing polymer is a polyethylene glycol.
 12. The analyte sensingcomposition of claim 1, wherein the sensing polymer is an enzymeselected from the group consisting of a glucose oxidase, a glucosedehydrogenase, a galactose oxidase, a glutamate oxidase, an L-amino acidoxidase, a D-amino acid oxidase, a cholesterol oxidase, a cholesterolesterase, a choline oxidase, a lipoxigenase, a lipoprotein lipase, aglycerol kinase, a glycerol-3-phosphate oxidase, a lactate oxidase, alactate dehydrogenase, a pyruvate oxidase, an alcohol oxidase, abilirubin oxidase, a sarcosine oxidase, a uricase (also called a urateoxidase), and an xanthine oxidase and wherein the analyte is a substratefor the enzyme.
 13. The analyte sensing composition of claim 1, whereinthe sensing polymer is a receptor protein and the analyte is a ligandwhich binds to the receptor.
 14. The analyte sensing composition ofclaim 13, wherein the receptor protein is a steroid receptor, or anestrogen receptor.
 15. The analyte sensing composition of claim 1,wherein the sensing protein is an enzyme which catalyzes oxidation orreduction of the analyte and wherein the sensing composition furthercomprises one or more redox mediators which function for electrontransfer from the analyte or the oxidation or reduction product thereofto the complexed carbon nanotube.
 16. The analyte sensing composition ofclaim 1, wherein the analyte is a steroid and the sensing polymer is asteroid receptor protein.
 17. The analyte sensing composition of claim16, wherein the analyte is a steroid and the sensing polymer iscovalently linked to one or more molecules of the steroid.
 18. Theanalyte sensing composition of claim 16, wherein the analyte is asteroid and the sensing polymer is covalently linked to one or moremolecules of an analog or derivative of the steroid.
 19. The analytesensing composition of claim 17, wherein the sensing composition furthercomprising a binding partner for the steroid.
 20. The analyte sensingcomposition of claim 17, wherein the sensing composition furthercomprises an antibody or antibody fragment that binds to the steroid.21. The analyte sensing composition of claim 16, wherein the steroid isan estrogen.
 22. The analyte sensing composition of claim 16, whereinthe steroid is 17-Beta-estradiol.
 23. The analyte sensing composition ofclaim 18, wherein the steroid is an estrogen, the sensing compositionfurther comprises a binding partner for the estrogen and the sensingpolymer is covalently linked to an analog or derivative of the estrogenthat binds to the estrogen binding partner.
 24. The analyte sensingcomposition of claim 18, wherein the steroid is an estrogen, the sensingcomposition further comprises an antibody or antibody fragment whichbinds to the estrogen and the sensing polymer is covalently linked to ananalog or derivative of the estrogen that binds to the antibody orantibody fragment.
 25. The analyte sensing composition of claim 1,wherein the analyte is a monosaccharide and the sensing polymer is apolysaccharide.
 26. The analyte sensing composition of claim 25, furthercomprising a sensing partner, wherein the monosaccharide and thepolysaccharide bind to the sensing partner.
 27. The analyte sensingcomposition of claim 26, wherein the analyte is glucose, thepolysaccharide comprises at least one glucose monomer and the sensingpartner is a protein that binds to the glucose and the at least oneglucose monomer.
 28. The analyte sensing composition of claim 27,wherein the polysaccharide is a dextran and the protein is concanavalinA or apo-glucose oxidase.
 29. A sensing element for detecting ananalyte, wherein the sensing element comprises: (a) the analyte sensingcomposition of claim 1, and (b) a selectively porous container forreceiving and retaining the analyte sensing composition, wherein thecontainer is sufficiently porous to allow the analyte to enter thecontainer without allowing the sensing complex of the sensing polymerwith the semi-conducting, single-walled carbon nanotube of the analytesensing composition to exit the container.
 30. A sensing system fordetecting one or more analytes, wherein the sensing system comprises:(a) one or more sensing elements of claim 29; (b) a source ofelectromagnetic radiation for exciting the photo-induced band gapfluorescence in the near-infrared of the carbon nanotube of the sensingcomplex; and (c) a detector for detecting the photo-induced band gapfluorescence of the carbon nanotube of the sensing complex.
 31. Thesensing system of claim 30, wherein the one or more sensing elements areimplanted within a mammal. 32.-37. (canceled) 38.-46. (canceled)
 47. Thecomposition of claim 25, wherein the monosaccharide is glucose.
 48. Thecomposition of claim 25, wherein the polysaccharide is a dextran.
 49. Asensing element for detecting an analyte, wherein the sensing elementcomprises: (a) the analyte sensing composition of claim 8, and (b) aselectively porous container for receiving and retaining the analytesensing composition, wherein the container is sufficiently porous toallow the analyte to enter the container without allowing the sensingcomplex of the polysaccharide with the semi-conducting, single-walledcarbon nanotube of the analyte sensing composition to exit thecontainer.
 50. A sensing element for detecting a monosaccharide, whereinthe sensing element comprises: (a) the analyte sensing composition ofclaim 25; and (b) a selectively porous container for receiving andretaining the analyte sensing composition, wherein the container issufficiently porous to allow the monosaccharide to enter the containerwithout allowing the sensing complex of the polysaccharide with thesemi-conducting, single-walled carbon nanotube of the analyte sensingcomposition to exit the container.
 51. A sensing system for detectingone or more analytes, wherein the sensing system comprises: (a) one ormore sensing elements for detecting an analyte of claim 49; (b) a sourceof electromagnetic radiation for exciting the photo-induced band gapfluorescence in the near-infrared of the carbon nanotube of the sensingcomplex; and (c) a detector for detecting the photo-induced band gapfluorescence in the near-infrared of the carbon nanotube of the sensingcomplex.
 52. The sensing system of claim 51, wherein the analyte isglucose.
 53. A sensing system for detecting one or more monosaccharides,wherein the sensing system comprises: (a) one or more sensing elementsfor detecting a monosaccharide of claim 50; (b) a source ofelectromagnetic radiation for exciting the photo-induced band gapfluorescence in the near infrared of the carbon nanotube of the sensingcomplex; and (c) a detector for detecting the photo-induced band gapfluorescence in the near-infrared of the carbon nanotube of the sensingcomplex.
 54. The sensing system of claim 53 wherein, the analyte isglucose.
 55. The analyte sensing composition of claim 1, wherein theanalyte sensing composition is dispersed in a liquid phase.
 56. Theanalyte sensing composition of claim 55, wherein the analyte sensingcomposition is dispersed in an aqueous medium.
 57. The analyte sensingcomposition of claim 1, wherein the analyte sensing composition isdispersed in a solid or semi-solid matrix wherein the matrix isselectively permeable to the analyte, but not permeable to the sensingcomplex.
 58. A method for detecting the presence of an analyte ordetermining the concentration of the analyte in an environment in whichthe analyte may be present, which comprises the steps of: (a) contactingthe analyte sensing composition of claim 1 with the environment in whichthe analyte may be present; and (b) detecting the photo-induced band gapfluorescence in the near-infrared of the carbon nanotube of the sensingcomposition to thereby detect the presence of the analyte or determinethe concentration of the analyte in the environment.
 59. The method ofclaim 58, wherein the analyte is a monosaccharide and the sensingpolymer of the sensing composition is a polysaccharide.
 60. The methodof claim 58, wherein the analyte sensing composition is retained in acontainer that is selectively porous to the analyte
 61. The method ofclaim 58, wherein the analyte sensing composition is dispersed in aliquid phase.
 62. The method of claim 61, wherein the analyte sensingcomposition is dispersed in an aqueous solution.
 63. The method of claim58, wherein the sensing composition is dispersed in a solid orsemi-solid matrix which is selectively permeable to the analyte, but notpermeable to the sensing complex of the analyte sensing composition. 64.An analyte sensing composition for detecting an analyte, or determiningthe concentration of the analyte, which consists essentially of asensing complex of a sensing polymer with a semi-conducting,single-walled carbon nanotube, wherein the carbon nanotube of thesensing complex exhibits photo-induced band gap fluorescence, thesensing polymer selectively binds to or selectively reacts with theanalyte, and the photo-induced band gap fluorescence of the carbonnanotube of the sensing complex is responsive to the selective bindingof the sensing polymer to the analyte or the selective reaction of thesensing polymer with the analyte.